Multivalent antibody complexes targeting IGF-1R show potent toxicity against solid tumors

ABSTRACT

The present invention concerns methods and compositions comprising an anti-IGF-1R antibody or fragment thereof for treatment of cancer or autoimmune disease. Preferably, the cancer is renal cell carcinoma, breast cancer or pancreatic cancer. The anti-IGF-1R antibody or fragment may be part of a complex, such as a DOCK-AND-LOCK™ (DNL™) (complex produced by binding interaction between anchor domain moiety of A-kinase anchoring protein and dimerization and docking domain moiety of protein kinase A regulatory subunit) complex. Preferably, the DNL™ (complex produced by binding interaction between anchor domain moiety of A-kinase anchoring protein and dimerization and docking domain moiety of protein kinase A regulatory subunit) complex also comprises a second antibody, a second antibody fragment, an affibody or a cytokine. More preferably, the cytokine is interferon-α2b. Most preferably, the second antibody, second fragment or affibody binds to IGF-1R, TROP2 or CEACAM6. The anti-IGF-1R antibody or complex may be administered alone or in combination with a therapeutic agent, such as an mTOR inhibitor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application Ser. No. 61/566,273, filed Dec. 2, 2011,and 61/616,051, filed Mar. 27, 2012. This application is a continuationof U.S. patent application Ser. No. 13/688,812, filed Nov. 29, 2012,which was a continuation-in-part of U.S. patent application Ser. No.13/483,761, filed May 30, 2012, which was a divisional of U.S. Ser. No.12/949,536, filed Nov. 18, 2010 (now U.S. Pat. No. 8,211,440), which wasa divisional of U.S. Ser. No. 12/396,605, filed Mar. 3, 2009 (now U.S.Pat. No. 7,858,070), which was a divisional of U.S. Ser. No. 11/633,729,filed Dec. 5, 2006 (now U.S. Pat. No. 7,527,787), which was acontinuation-in-part of PCT/US06/10762, filed Mar. 24, 2006, and acontinuation-in-part of PCT/US06/12084, filed Mar. 29, 2006, and acontinuation-in-part of PCT/US06/25499, filed Jun. 29, 2006, and acontinuation-in-part of U.S. Ser. No. 11/389,358, filed Mar. 24, 2006(now U.S. Pat. No. 7,550,143), and a continuation-in-part of U.S. Ser.No. 11/391,584, filed Mar. 28, 2006 (now U.S. Pat. No. 7,521,056), and acontinuation-in-part of U.S. Ser. No. 11/478,021, filed Jun. 29, 2006(now U.S. Pat. No. 7,534,866), and which claimed the benefit under 35U.S.C. 119(e) of provisional U.S. Patent Applicant Ser. No. 60/782,332,filed Mar. 14, 2006, and 60/751,196, filed Dec. 16, 2005, and60/728,292, filed Oct. 19, 2005. U.S. Ser. No. 11/633,729 claimed thebenefit under 35 U.S.C. 119(e) of provisional U.S. Patent ApplicationSer. No. 60/751,196, filed Dec. 16, 2005, and 60/864,530, filed Nov. 6,2006. This application is also a continuation-in-part of U.S. patentapplication Ser. No. 12/722,645, filed Mar. 12, 2010, which was acontinuation-in-part of U.S. Ser. No. 12/689,336, filed Jan. 19, 2010,which claimed the benefit under 35 U.S.C. 119(e) of provisional U.S.Patent Application Ser. No. 61/145,896, filed Jan. 20, 2009. Eachpriority application is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 26, 2012, isnamed IBC134US1.txt and is 54,756 bytes in size.

FIELD

The present invention relates to compositions and methods of use ofmultivalent, complexes, preferably multivalent, multispecific complexes,more preferably multivalent, bispecific complexes, that comprise one ormore antibodies or antigen-binding fragments thereof that bind to theinsulin-like growth factor type I receptor (IGF-1R), but not to theinsulin receptor (IR). In other preferred embodiments, the multivalentcomplexes comprise a second antibody or antigen-binding fragment thereofthat binds to a different tumor-associated antigen (TAA), such as TROP2or CEACAM6. In alternative embodiments, the complexes may comprise acytokine, such as interferon-α2b. In most preferred embodiments, themultivalent complex is a DOCK-AND-LOCK™ (DNL™) (complex produced bybinding interaction between anchor domain moiety of A-kinase anchoringprotein and dimerization and docking domain moiety of protein kinase Aregulatory subunit) complex. The complex may be administered to asubject, preferably a human subject, for treatment of a disease ormedical condition. Preferably the disease is cancer, more preferablyrenal cell carcinoma, breast cancer or pancreatic cancer. However, theskilled artisan will realize that other forms of cancer which expressIGF-1R may also be treated. The complexes may be administered alone orin combination with one or more therapeutic agents administered before,simultaneously with, or after the complex. In a particular embodiment,the complex exhibits a synergistic effect with a therapeutic agent, suchas an mTOR inhibitor.

BACKGROUND

The insulin-like growth factor type I receptor (IGF-1R) is a member ofthe large class of tyrosine kinase receptors, which regulate a varietyof intracellular pathways. IGF-1R binds IGF-1, a polypeptide hormonestructurally similar to insulin (Laron, Mol Pathol. 2001, 54:311-16).The IGF-1 receptor is homologous to the insulin receptor (IR), sharingabout 70% overall sequence homology with IR (Riedemann and Macaulay,Endocrine-Related Cancer, 2006, 13:S33-43). Not surprisingly, inhibitorsdeveloped against IGF-1R tend to show cross-reactivity with the insulinreceptor, accounting for at least part of the toxicity profiles of suchcompounds (Miller and Yee, 2005, Cancer Res. 65:10123-27; Riedemann andMacaulay, 2006).

The IGF system plays an important role in regulating cell proliferation,differentiation, apoptosis and transformation (Jones et al,Endocrinology Rev. 1995. 16:3-34). The IGF system comprises tworeceptors, insulin like growth factor receptor 1 (IGF-1R; CD221) andinsulin like growth factor receptor 2 (IGF-2R; CD222); two ligands,insulin like growth factor 1 (IGF-1) and IGF-2; and several IGF bindingproteins (IGFBP-1 to IGFBP-6). In addition, a large group of IGFBPproteases (e.g., caspases, metalloproteinases, prostate-specificantigen) hydrolyze IGF bound IGFBP to release free IGFs, which theninteract with IGF-1R and IGF-2R.

IGF-1R comprises two extracellular a subunits (130-135 kD) and twomembrane spanning β-subunits (95 kD) that contain the cytoplasmictyrosine kinase domain. IGF-1R, like the insulin receptor (IR), differsfrom other receptor tyrosine kinase family members by having a covalentdimeric (α2β2) structure. IGF-1R contains 84% sequence identity to IR inthe kinase domain, while the membrane and C-terminal regions share 61%and 44% sequence identity, respectively (Ulrich et al., EMBO J., 1986,5:2503-12; Blakesley et al., Cytokine Growth Factor Rev., 1996.7:153-56).

IGF-1 and IGF-2 are activating ligands of IGF-1R. Binding of IGF-1 andIGF-2 to the α-chain induces conformational changes that result inautophosphorylation of each β-chain at specific tyrosine residues,converting the receptor from the unphosphorylated inactive state to thephosphorylated active state. The activation of three tyrosine residuesin the activation loop (Tyr residues at 1131, 1135 and 1136) of thekinase domain leads to an increase in catalytic activity that triggersdocking and phosphorylation of substrates such as IRS-1 and Shc adaptorproteins. Activation of these substrates leads to phosphorylation ofadditional proteins involved in the signaling cascade of survival (PI3K,AKT, TOR, S6) and/or proliferation (mitogen-activated protein kinase,p42/p44) (Pollak et al., Nature Reviews Cancer. 2004. 4:505-516; Basergaet al., Biochim Biophys Acta. 1997. 1332:F105-F126; Baserga et al, Int.J. Cancer. 2003. 107:873-77).

IGF-1R has anti-apoptotic effects in both normal and cancer cells(Resnicoff et al., 1995, Cancer Res. 55:2463-69; Kang et al., Am JPhysiol Renal Physiol., 2003, 285:F1013-24; Riedemann and Macaulay,2006). IGF-1R activation has been reported to be significant in thedevelopment of resistance to a variety of cytotoxic agents, such aschemotherapeutic agents, radionuclides and EGFR inhibitors (Jones etal., Endocr Relat Cancer 2004, 11:793-814; Warshamana-Greene et al.,2005, Clin. Cancer Res. 11:1563-71; Riedemann and Macaulay, 2006; Lloretet al., 2007, Gynecol. Oncol. 106:8-11). IGF-1R is overexpressed in awide range of tumor lines, such as melanoma, neuroblastoma, coloncancer, prostate cancer, renal cancer, breast cancer and pancreaticcancer (Ellis et al., 1998, Breast Cancer Treat. 52:175-84; van Golen etal., 2000, Cell Death Differ. 7:654-65; Zhang et al., 2001, BreastCancer Res. 2:170-75; Jones et al., 2004; Riedemann and Macaulay, 2006).A functional IGF-1R is required for transformation and promotes cancercell growth, survival and metastasis (Riedemann and Macaulay, 2006).

Attempts have been made to develop IGF-1R inhibitors for use asanti-cancer agents, such as tyrphostins, pyrrolo[2,3-d]-pyrimidinederivatives, nordihydroguaiaretic acid analogs, diaryureas, AG538,AG1024, NVP-AEW541, NVP-ADW742, BMS-5326924, BMS-554417, OSI-906,INSM-18, luteolin, simvastatin, silibinin, black tea polyphenols,picropodophyllin, anti-IGF-1R antibodies and siRNA inhibitors (Arteagaet al., 1998, J Clin Invest. 84:1418-23; Warshamana-Greene et al., 2005;Klein and Fischer, 2002, Carcinogenesis 23:217-21; Blum et al., 2000,Biochemistry 39:15705-12; Garcia-Echeverria et al., 2004, Cancer Cell5:231-39; Garber, 2005, JNCI 97:790-92; Bell et al., 2005, Biochemistry44:930-40; Wu et al., 2005, Clin Cancer Res 11:3065-74; Wang et al.,2005, Mol Cancer Ther 4:1214-21; Singh and Agarwal, 2006, Mol Carinog.45:436-42; Gable et al., 2006, Mol Cancer Ther 5:1079-86; Niu et al.,Cell Biol Int., 2007, 31:156-64; Blecha et al., 2007, Biorg Med ChemLett. 17:4026-29; Qian et al., 2007, Acta Biochim Biophys Sin,39:137-47; Fang et al., 2007, Carcinogenesis 28:713-23; Cohen et al.,2005, Clin Cancer Res 11:2063-73; Sekine et al., Biochem Biophys ResCommun., 2008, 25:356-61; Haluska et al., 2008, J Clin Oncol. 26: May 20suppl; abstr 14510; U.S. Patent Application Publ. No. 2006-233810, theExamples section of each of which is incorporated herein by reference).Typically, these agents have tended to cross-react to a greater orlesser extent with both IGF-1R and IR and/or to act as IGF-1R agonists.The use of such agents for cancer therapy has been limited by theirtoxicity (Riedemann and Macaulay, 2006). A need exists in the field formore effective forms of anti-IGF-1R antibodies and complexes thereof.

SUMMARY

The present invention concerns compositions and methods of use ofmultivalent complexes comprising anti-IGF-1R antibodies or fragmentsthereof. The complexes may further comprise a second anti-TAA antibodyor fragment thereof, such as an anti-TROP2 or anti-CEACAM6 antibody orantibody fragment. Alternatively, the complexes may comprise a cytokine,such as interferon-α2b. Preferably, the complex comprising two differentantibodies, or an antibody and a cytokine, have greater activity thanthe individual antibodies alone, the individual antibody or individualcytokine, or the combination of unconjugated antibodies or unconjugatedantibody and unconjugated cytokine.

Preferably, the anti-IGF-1R antibodies bind to IGF-1R but not to IR.More preferably, the anti-IGF-1R antibodies are not agonists of IGF-1R.Most preferably, the anti-IGF-1R antibodies bind to an epitope of IGF-1Rcomprising the first half of the cysteine-rich domain of IGF-1R, betweenamino acid residues 151 and 222 of the human IGF-1R sequence. (See,e.g., Adams et al., Cell Mol Life Sci 57:1050-93, 2000; NCBI AccessionNo. AAB22215).

In certain embodiments, the anti-IGF-1R antibody is a murine, chimeric,humanized or human antibody or antigen-binding fragment thereofcomprising the heavy chain CDR sequences CDR1 (DYYMY, SEQ ID NO:85),CDR2 (YITNYGGSTYYPDTVKG, SEQ ID NO:86) and CDR3 (QSNYDYDGWFAY, SEQ IDNO:87) and the light chain CDR sequences CDR1 (KASQEVGTAVA, SEQ IDNO:88), CDR2 (WASTRHT, SEQ ID NO:89) and CDR3 (QQYSNYPLT, SEQ ID NO:90).In alternative embodiments, the anti-IGF-1R antibody is a chimeric,humanized or human antibody that binds to the same epitope of IGF-1Rand/or that blocks binding to IGF-1R of a murine R1 antibody comprisingthe heavy chain CDR sequences CDR1 (DYYMY, SEQ ID NO:85), CDR2(YITNYGGSTYYPDTVKG, SEQ ID NO:86) and CDR3 (QSNYDYDGWFAY, SEQ ID NO:87)and the light chain CDR sequences CDR1 (KASQEVGTAVA, SEQ ID NO:88), CDR2(WASTRHT, SEQ ID NO:89) and CDR3 (QQYSNYPLT, SEQ ID NO:90). Theanti-IGF-1R antibody may be a naked antibody or may be animmunoconjugate attached to at least one therapeutic agent and/or atleast one diagnostic agent.

Although the second anti-TAA antibody or fragment thereof may be ananti-TROP2 or anti-CEACAM6 antibody or fragment, in alternativeembodiments the second antibody or fragment may bind to any of a numberof known tumor-associated antigens, such as carbonic anhydrase IX,CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29,CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95,CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7,ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB,HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growthfactor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4,MUC5ac, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR,tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2),VEGFR, EGFR, PlGF, complement factors C3, C3a, C3b, C5a, C5, and anoncogene product.

The second anti-TAA antibody may be selected from any of a wide varietyof anti-cancer antibodies known in the art, including but not limited tohPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,151,164), hA19(U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1 (U.S.Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat.No. 7,387,772), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No.6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. ProvisionalPatent Application 61/145,896), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections. In certain embodiments, a second, differentanti-IGF-1R antibody may be used, such as any of the anti-IGF-1Rantibodies in clinical development (see, e.g., Ryan and Goss, TheOncologist, 2008, 13:16-24).

In various embodiments, the complexes may be DOCK-AND-LOCK™ (DNL™)complexes. The technology to make DNL™ complexes has been described inU.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400;7,906,118; 8,003,111 and 8,034,352, the Examples section of eachincorporated herein by reference. The technique relies upon the bindinginteraction between a dimerization and docking domain (DDD) moiety ofhuman protein kinase A (PKA) regulatory subunit RIα, RIβ, RIIα or RIIβand an anchor domain (AD) moiety of an A-kinase anchoring protein(AKAP). The PKA DDD moieties spontaneously form dimers that bind to anAD moiety to join the complex together. The AD and DDD moiety may beattached to an effector, such as an antibody, antibody fragment,cytokine, toxin, enzyme, hormone or other protein or peptide, forexample in the form of a fusion protein. Alternatively, the AD and DDDmoieties may be attached to effectors by other covalent linkage, such asby chemical cross-linking. The technique is not limiting and anyeffector moiety that may be attached to an AD or DDD moiety may beincorporated into a DNL™ complex.

The anti-IGF-1R containing complex may be administered alone, or incombination with one or more therapeutic agents. The agents may beattached to the complex or may be administered separately. As discussedbelow, therapeutic agents may include, but are not limited to,radionuclides, immunomodulators, anti-angiogenic agents, cytokines,chemokines, growth factors, hormones, drugs, prodrugs, enzymes,oligonucleotides, siRNAs, pro-apoptotic agents, photoactive therapeuticagents, cytotoxic agents, chemotherapeutic agents, toxins, otherantibodies or antigen binding fragments thereof. In preferredembodiments the therapeutic agent may be an EGFR inhibitor (e.g.,erlotinib or anti-EGFR antibody, such as ERBITUX® (cetuximab)), anIGF-1R inhibitor such as tryphostins (e.g., AG1024, AG538),pyrrolo[2,3-d]-pyrimidine derivatives (e.g., NVP-AEW541) or an mTORinhibitor such as temsirolimus, rapamycin, ridaforolimus or everolimus.

Any cancer or diseased cell that expresses IGF-1R may be treated and/ordiagnosed with the anti-IGF-1R antibodies, including but not limited toWilms' tumor, Ewing sarcoma, neuroendocrine tumors, glioblastomas,neuroblastoma, melanoma, skin, breast, colon, rectum, prostate, liver,renal, pancreatic and/or lung cancer, as well as lymphomas, leukemias,and myelomas. Other forms of cancer that may be treated include but arenot limited to acute lymphoblastic leukemia, acute myelogenous leukemia,biliary cancer, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, endometrial cancer, esophageal cancer, gastriccancer, head and neck cancer, Hodgkin's lymphoma, medullary thyroidcarcinoma, non-Hodgkin's lymphoma, ovarian cancer, glioma and urinarybladder cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1A. Anti-IGF-1R mediated growth inhibition under serum-freeconditions. Caki-2 cells were grown in serum-free media containingholo-transferrin (10 mg/mL) for 24 h before the addition of theantibodies. After a 1-h incubation with the antibodies, IGF-1 (100ng/mL) was added to all the wells. Plates were incubated for a further96 h before MTS reagent was added and cell growth inhibition determinedusing Prism Graph Pad software.

FIG. 1B. Anti-IGF-1R mediated growth inhibition under serum-freeconditions. ACHN cells were grown in serum-free media containingholo-transferrin (10 mg/mL) for 24 h before the addition of theantibodies. After a 1-h incubation with the antibodies, IGF-1 (100ng/mL) was added to all the wells. Plates were incubated for a further96 h before MTS reagent was added and cell growth inhibition determinedusing Prism Graph Pad software.

FIG. 1C. Anti-IGF-1R mediated growth inhibition under serum-freeconditions. 786-0 cells were grown in serum-free media containingholo-transferrin (10 mg/mL) for 24 h before the addition of theantibodies. After a 1-h incubation with the antibodies, IGF-1 (100ng/mL) was added to all the wells. Plates were incubated for a further96 h before MTS reagent was added and cell growth inhibition determinedusing Prism Graph Pad software.

FIG. 2A. Characterization of 1R-2b in RCC. Based on the luciferasereporter gene assay (iLite kit), 1R-2b yielded a specific activity of15×10⁶ U/mg or 3750 U/pmole versus 180 and 3255 U/pmole for twodifferent pegylated-IFN molecules.

FIG. 2B. Characterization of 1R-2b in RCC. In growth inhibition assaysof 786-0 cells, 1R-2b had an EC50 values of 49 pm.

FIG. 2C. Characterization of 1R-2b in RCC. In growth inhibition assaysof ACHN cells, 1R-2b had an EC50 value 62 pM.

FIG. 3A. Growth inhibition under anchorage-independent conditions. A 1%base agar was mixed 1:1 with 2× growth media (10% FBS finalconcentration) and added to wells of a 24-well plate. ACHN cells in 2×growth media were mixed 1:1 with 0.7% agarose and added (1250 cells perwell) to the base agar. Cells were fed by weekly replacement of growthmedia on the top of the agarose layer. Treated wells contained the testarticles in the agarose/cell layer at the beginning and in subsequentfeedings. Once colonies were clearly visible by microscopy in untreatedcontrol wells, the medium was removed and the colonies stained withcrystal violet. Colonies were counted under a microscope and the averagenumber was determined from five different fields of view within thewell.

FIG. 3B. Growth inhibition under anchorage-independent conditions.Conditions were as disclosed in the legend to FIG. 3A, with theexception that 786-0 cells were used.

FIG. 4A. Synergy between anti-IGF-1R treatment and an mTOR inhibitor.ACHN cells were harvested, washed in PBS several times to remove FBS,and plated in 96-wells plates overnight in SFM. On the following day,various doses (1 mM to 0.06 nM) of the mTOR inhibitor temsirolimus wasadded to the plates with and without hR1 or Hex-hR1 (100, 10, and 1 nMconstant amounts) or 1R-2b (26, 2.6, or 0.26 nM; NOTE: 26 nM 1R-2b˜100,000 Units/mL of IFN). IGF-1 was added at 100 ng/mL. Plates wereincubated for 96-h before MTS substrate was added to all the wells andthe plates read at 492 nm. Data was graphed as Percent Growth Inhibitionvs. [temsirolimus]. IC50-values for temsirolimus were determined foreach condition and Combinatorial Index (CI) was calculated based onchanges in these values when co-incubated with hR1, Hex-hR1, or 1R-2b(CI<1 for synergy). Combination of temsirolimus with hR1 (CI=0.64). TheIC₅₀ values for temsirolimus concentration needed to mediate 50%inhibition of cell growth were 7.76 nM for Tem alone (R² 0.94); 1.45 nMwith 100 nM hR1 (R² 0.88); 0.56 nM with 10 nM hR1 (R² 0.84); and 2.86 nMwith 1 nM hR1 (R² 0.93). Synergy with an mTOR inhibitor occurred atconcentrations as low as 10 nM.

FIG. 4B. Synergy between anti-IGF-1R treatment and an mTOR inhibitor.Conditions were as disclosed in the legend to FIG. 4A. Combination oftemsirolimus with Hex-hR1 (CI=0.43). The IC₅₀ values were 7.76 nM forTem alone (R² 0.94); 3.15 nM with 1 nM Hex-hR1 (R² 0.63); 0.06 nM with10 nM Hex-hR1 (R² 0.66); and <0.06 nM with 100 nM HexhR1 (R² 0.63).Synergy with an mTOR inhibitor occurred at concentrations as low as 1nM.

FIG. 4C. Synergy between anti-IGF-1R treatment and an mTOR inhibitor.Conditions were as disclosed in the legend to FIG. 4A. Combination oftemsirolimus with 1R-2b (CI=0.02). The IC₅₀ values were 7.76 nM for Temalone (R² 0.94); <0.06 nM with 26 nM 1R-2b (R² 0.32); <0.06 nM with 2.6nM 1R-2b (R² 0.34); and 12.7 nM with 0.26 nM 1R-2b (R² 0.81). Synergywith an mTOR inhibitor occurred at concentrations as low as 2.6 nM.

DETAILED DESCRIPTION Definitions

Unless otherwise specified, “a” or “an” means “one or more”.

As used herein, the term “about” means plus or minus ten percent (10%)of a value. For example, “about 100” would refer to any number between90 and 110.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, peptides, drugs, toxins, enzymes,nucleases, hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,oligonucleotides (e.g., RNAi or siRNA) and radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), contrast agents, fluorescent compounds or molecules, andenhancing agents (e.g., paramagnetic ions) for magnetic resonanceimaging (MRI).

An “antibody” as used herein refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. An“antibody” includes monoclonal, polyclonal, bispecific, multispecific,murine, chimeric, humanized and human antibodies.

A “naked antibody” is an antibody or antigen binding fragment thereofthat is not attached to a therapeutic or diagnostic agent. The Fcportion of an intact naked antibody can provide effector functions, suchas complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev50:59-87, 1998). Other mechanisms by which naked antibodies induce celldeath may include apoptosis. (Vaswani and Hamilton, Ann Allergy AsthmaImmunol 81: 105-119, 1998.)

An “antibody fragment” is a portion of an intact antibody such asF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv, scFv, dAb and the like. Regardlessof structure, an antibody fragment binds with the same antigen that isrecognized by the full-length antibody. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains or recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). “Single-chain antibodies”, often abbreviated as“scFv” consist of a polypeptide chain that comprises both a V_(H) and aV_(L) domain which interact to form an antigen-binding site. The V_(H)and V_(L) domains are usually linked by a peptide of 1 to 25 amino acidresidues. Antibody fragments also include diabodies, triabodies andsingle domain antibodies (dAb).

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains. Additional FR amino acidsubstitutions from the parent, e.g. murine, antibody may be made. Theconstant domains of the antibody molecule are derived from those of ahuman antibody.

A “human antibody” is an antibody obtained from transgenic mice thathave been genetically engineered to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. (See, e.g., McCafferty et al., Nature 348:552-553 (1990) forthe production of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). Human antibodies may also be generated by in vitro activated Bcells. (See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

Antibodies and Antibody Fragments

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

Antibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized antibodies are well known in the art(see, e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al.,Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988),Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit.Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844(1993)). A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXENOMOUSE® (mouse genetically engineered to produce human antibodies)(e.g., Green et al., 1999, J. Immunol. Methods 231:11-23) from Abgenix(Fremont, Calif.). In the XENOMOUSE® (mouse genetically engineered toproduce human antibodies) and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XenoMouse® (mouse genetically engineered to produce humanantibodies) was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®(mouse genetically engineered to produce human antibodies) immunizedwith a target antigen will produce human antibodies by the normal immuneresponse, which may be harvested and/or produced by standard techniquesdiscussed above. A variety of strains of XenoMouse® (mouse geneticallyengineered to produce human antibodies) are available, each of which iscapable of producing a different class of antibody. Transgenicallyproduced human antibodies have been shown to have therapeutic potential,while retaining the pharmacokinetic properties of normal humanantibodies (Green et al., 1999). The skilled artisan will realize thatthe claimed compositions and methods are not limited to use of theXenoMouse® (mouse genetically engineered to produce human antibodies)system but may utilize any transgenic animal that has been geneticallyengineered to produce human antibodies.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

Techniques for producing single domain antibodies (DABs) are also knownin the art, as disclosed for example in Cossins et al. (2006, ProtExpress Purif 51:253-259), incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Known Antibodies

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040; 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art (see, e.g., U.S. Pat. Nos. 7,531,327; 7,537,930;7,608,425 and 7,785,880, the Examples section of each of which isincorporated herein by reference). Such known antibodies may be used incombination with one or more anti-IGF-1R antibodies, either as part of acomplex, or administered separately or together with an anti-IGF-1Rantibody.

Particular antibodies that may be of use for therapy of cancer withinthe scope of the claimed methods and compositions include, but are notlimited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7(anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX),hL243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),cetuxiamab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan(anti-CD20); panitumumab (anti-EGFR); rituximab (anti-CD20); tositumomab(anti-CD20); GA101 (anti-CD20); and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20040202666(now abandoned); 20050271671; and 20060193865; the Examples section ofeach incorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE). While anti-IGF-1R antibodies haveprimarily been addressed to cancer therapy to date, there areindications that IGF-1R may also be involved in immune system functionand autoimmune diseases (see, e.g., Smith, 2010, Pharm Rev 62:199-236).

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab), CD74 (milatuzumab), CD19(hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see, e.g., Winer et al.,2011, Nature Med 17:610-18). Anti-CD3 antibodies also have been proposedfor therapy of type 1 diabetes (Cernea et al., 2010, Diabetes Metab Rev26:602-05).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Bapineuzumab is in clinical trials for Alzheimer's disease therapy.Other antibodies proposed for therapy of Alzheimer's disease include Alz50 (Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,and solanezumab. Infliximab, an anti-TNF-α antibody, has been reportedto reduce amyloid plaques and improve cognition.

Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and inclinical trials as imaging agents for disclosing said clots andpulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL), anti-MIF, and anti-CD74 (e.g., hLL1) antibodies canbe used to target atherosclerotic plaques. Abciximab (anti-glycoproteinIIb/IIIa) has been approved for adjuvant use for prevention ofrestenosis in percutaneous coronary interventions and the treatment ofunstable angina (Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3antibodies have been reported to reduce development and progression ofatherosclerosis (Steffens et al., 2006, Circulation 114:1977-84).Antibodies against oxidized LDL induced a regression of establishedatherosclerosis in a mouse model (Ginsberg, 2007, J Am Coll Cardiol52:2319-21). Anti-ICAM-1 antibody was shown to reduce ischemic celldamage after cerebral artery occlusion in rats (Zhang et al., 1994,Neurology 44:1747-51). Commercially available monoclonal antibodies toleukocyte antigens are represented by: OKT anti-T-cell monoclonalantibodies (available from Ortho Pharmaceutical Company) which bind tonormal T-lymphocytes; the monoclonal antibodies produced by thehybridomas having the ATCC accession numbers HB44, HB55, HB12, HB78 andHB2; G7Ell, W8E7, NKP15 and GO22 (Becton Dickinson); NEN9.4 (New EnglandNuclear); and FMCll (Sera Labs). A description of antibodies againstfibrin and platelet antigens is contained in Knight, Semin. Nucl. Med.,20:52-67 (1990).

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:120) and veltuzumab (SEQ IDNO:121).

Rituimab heavy chain variable region sequence  (SEQ ID NO: 120)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVeltuzumab heavy chain variable region  (SEQ ID NO: 121)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes 214 356/358 431 Complete allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Immunoconjugates

In certain embodiments, the antibodies or fragments thereof may beconjugated to one or more therapeutic or diagnostic agents. Thetherapeutic agents do not need to be the same but can be different, e.g.a drug and a radioisotope. For example, ¹³¹I can be incorporated into atyrosine of an antibody or fusion protein and a drug attached to anepsilon amino group of a lysine residue. Therapeutic and diagnosticagents also can be attached, for example to reduced SH groups and/or tocarbohydrate side chains. Many methods for making covalent ornon-covalent conjugates of therapeutic or diagnostic agents withantibodies or fusion proteins are known in the art and any such knownmethod may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

An alternative method for attaching toxins or other functional groups toantibodies or complexes thereof involves use of click chemistryreactions. The click chemistry approach was originally conceived as amethod to rapidly generate complex substances by joining small subunitstogether in a modular fashion. (See, e.g., Kolb et al., 2004, Angew ChemInt Ed 40:3004-31; Evans, 2007, Aust J Chem 60:384-95.) Various forms ofclick chemistry reaction are known in the art, such as the Huisgen1,3-dipolar cycloaddition copper catalyzed reaction (Tornoe et al.,2002, J Organic Chem 67:3057-64), which is often referred to as the“click reaction.” Other alternatives include cycloaddition reactionssuch as the Diels-Alder, nucleophilic substitution reactions (especiallyto small strained rings like epoxy and aziridine compounds), carbonylchemistry formation of urea compounds and reactions involvingcarbon-carbon double bonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is a 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted akyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.)

Bispecific and Multispecific Antibodies

Certain embodiments may involve bispecific or even multispecificcomplexes comprising an anti-IGF-1R antibody or antibody fragment.Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of eachincorporated herein by reference. Reduction of the peptide linker lengthto less than 12 amino acid residues prevents pairing of V_(H) and V_(L)domains on the same chain and forces pairing of V_(H) and V_(L) domainswith complementary domains on other chains, resulting in the formationof functional multimers. Polypeptide chains of V_(H) and V_(L) domainsthat are joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are favored, but the exact patterns of oligomerization appearto depend on the composition as well as the orientation of V-domains(V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to the linkerlength.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.A more recent technique to produce DNL™ complexes, described in moredetail below, has been utilized to produce combinations of virtually anydesired antibodies, antibody fragments and other effector molecules. Thetechnique allows the assembly of monospecific, bispecific ormultispecific antibodies, either as naked antibody moieties or incombination with a wide range of other effector molecules such asimmunomodulators, enzymes, chemotherapeutic agents, chemokines,cytokines, diagnostic agents, therapeutic agents, radionuclides, imagingagents, anti-angiogenic agents, growth factors, oligonucleotides,hormones, peptides, toxins, pro-apoptotic agents, or a combinationthereof. Any of the techniques known in the art for making bispecific ormultispecific antibodies may be utilized in the practice of thepresently claimed methods.

Affibodies and Fynomers

Certain alternative embodiments may utilize affibodies in place ofantibodies. Affibodies are commercially available from AFFIBODY® (smallprotein antibody mimetic) AB (Solna, Sweden). Affibodies are smallproteins that function as antibody mimetics and are of use in bindingtarget molecules. Affibodies were developed by combinatorial engineeringon an alpha helical protein scaffold (Nord et al., 1995, Protein Eng8:601-8; Nord et al., 1997, Nat Biotechnol 15:772-77). The AFFIBODY®(small protein antibody mimetic) design is based on a three helix bundlestructure comprising the IgG binding domain of protein A (Nord et al.,1995; 1997). Affibodies with a wide range of binding affinities may beproduced by randomization of thirteen amino acids involved in the Fcbinding activity of the bacterial protein A (Nord et al., 1995; 1997).After randomization, the PCR amplified library was cloned into aphagemid vector for screening by phage display of the mutant proteins.The phage display library may be screened against any known antigen,using standard phage display screening techniques (e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, Quart. J. Nucl.Med. 43:159-162), in order to identify one or more affibodies againstthe target antigen.

A ¹⁷⁷Lu-labeled AFFIBODY® specific for HER2/neu has been demonstrated totarget HER2-expressing xenografts in vivo (Tolmachev et al., 2007,Cancer Res 67:2773-82). Although renal toxicity due to accumulation ofthe low molecular weight radiolabeled compound was initially a problem,reversible binding to albumin reduced renal accumulation, enablingradionuclide-based therapy with labeled AFFIBODY® (Id.).

The feasibility of using radiolabeled AFFIBODY® molecules for in vivotumor imaging has been recently demonstrated (Tolmachev et al., 2011,Bioconjugate Chem 22:894-902). A maleimide-derivatized NOTA wasconjugated to the anti-HER2 AFFIBODY® and radiolabeled with ¹¹¹In (Id.).Administration to mice bearing the HER2-expressing DU-145 xenograft,followed by gamma camera imaging, allowed visualization of the xenograft(Id.).

Fynomers can also bind to target antigens with a similar affinity andspecificity to antibodies. Fynomers are based on the human Fyn SH3domain as a scaffold for assembly of binding molecules. The Fyn SH3domain is a fully human, 63 amino acid protein that can be produced inbacteria with high yields. Fynomers may be linked together to yield amultispecific binding protein with affinities for two or more differentantigen targets. Fynomers are commercially available from COVAGEN AG(Zurich, Switzerland).

The skilled artisan will realize that AFFIBODY® molecules or fynomersmay be used as targeting molecules in the practice of the claimedmethods and compositions.

Pre-Targeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other therapeutic agent isattached to a small delivery molecule (targetable construct ortargetable construct) that is cleared within minutes from the blood. Apre-targeting bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702;6,962,702; 7,074,405; and U.S. Ser. No. 10/114,315 (now abandoned); theExamples section of each of which is incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents. The technique mayalso be utilized for antibody dependent enzyme prodrug therapy (ADEPT)by administering an enzyme conjugated to a targetable construct,followed by a prodrug that is converted into active form by the enzyme.

DOCK-AND-LOCK™ (DNL™)

In preferred embodiments, an anti-IGF-1R complex is formed as aDOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section ofeach of which is incorporated herein by reference.) Generally, thetechnique takes advantage of the specific and high-affinity bindinginteractions that occur between a dimerization and docking domain (DDD)sequence of the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and an anchor domain (AD) sequence derived from any of a varietyof AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wongand Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and ADpeptides may be attached to any protein, peptide or other molecule.Because the DDD sequences spontaneously dimerize and bind to the ADsequence, the technique allows the formation of complexes between anyselected molecules that may be attached to DDD or AD sequences.

Although the standard DNL™ complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL™complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL™ complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has a and 13 isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL™constructs of different stoichiometry may be produced and used (see,e.g., U.S. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL™construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA  DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA  AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA  AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC 

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKE EAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC 

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL™ complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKE EAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEE NRQILA PKA RIIα(SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ  PKA RIIβ(SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER 

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA 

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 2 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 91. S H I Q I P P G L T E L LQ G Y T V E V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F TR L R E A R A N N E D L D S K K D L K L I I I V V VTHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 12)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA 

TABLE 3 Conservative Amino Acid Substitutions in AD1 (SEQ ID NO: 3).Consensus sequence disclosed as SEQ ID NO: 92. Q I E Y L A K Q I V D N AI Q Q A N L D F I R N E Q N N L V T V I S VNIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA (SEQ ID NO: 37)QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA (SEQ ID NO: 41)QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA (SEQ ID NO: 43)QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA (SEQ ID NO: 45)QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV (SEQ ID NO: 49)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL™ constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQA Alternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA (SEQ ID NO: 52) QIEYHAKQIVDHAIHQA  (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA 

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 55) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 57)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 59) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP7(SEQ ID NO: 60) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 61)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 62) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 63) LAWKIAKMIVSDVMQQ 

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY  RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE  PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC 

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA 

Can et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A 

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1). Consensussequence disclosed as SEQ ID NO: 93. S H I Q I P P G L T E L L Q G Y T VE V L R T N S I L A Q  Q P P D L V E F A V E Y F T R L R E A R A N I D SK K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL™ constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Therapeutic Agents

In various embodiments, therapeutic agents such as cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes orother agents may be used, either conjugated to the subject anti-IGF-1Rcomplexes or separately administered before, simultaneously with, orafter the anti-IGF-1R complex. Drugs of use may possess a pharmaceuticalproperty selected from the group consisting of antimitotic, antikinase,alkylating, antimetabolite, antibiotic, alkaloid, anti-angiogenic,pro-apoptotic agents and combinations thereof.

Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine,anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,vinblastine, vincristine and vinca alkaloids.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, an hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT.

Radionuclides of use include, but are not limited to—¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Ru, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclidepreferably has a decay-energy in the range of 20 to 6,000 keV,preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV. Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like. Someuseful diagnostic nuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or ¹¹¹In.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2 or p53. Apreferred form of therapeutic oligonucleotide is siRNA.

Diagnostic Agents

Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹¹⁰In,¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III). Ultrasound contrast agents may comprise liposomes, suchas gas filled liposomes. Radiopaque diagnostic agents may be selectedfrom compounds, barium compounds, gallium compounds, and thalliumcompounds. A wide variety of fluorescent labels are known in the art,including but not limited to fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. Chemiluminescent labels of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt or an oxalate ester.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,such as a mammal, including humans, domestic or companion pets, such asdogs and cats, comprising administering to the subject a therapeuticallyeffective amount of a multivalent complex comprising an anti-IGF-1Rantibody or fragment.

In one embodiment, immunological diseases which may be treated with thesubject anti-IGF-1R complexes may include, for example, joint diseasessuch as ankylosing spondylitis, juvenile rheumatoid arthritis,rheumatoid arthritis; neurological disease such as multiple sclerosisand myasthenia gravis; pancreatic disease such as diabetes, especiallyjuvenile onset diabetes; gastrointestinal tract disease such as chronicactive hepatitis, celiac disease, ulcerative colitis, Crohn's disease,pernicious anemia; skin diseases such as psoriasis or scleroderma;allergic diseases such as asthma and in transplantation relatedconditions such as graft versus host disease and allograft rejection.

The administration of the anti-IGF-1R complexes can be supplemented byadministering concurrently or sequentially a therapeutically effectiveamount of another antibody that binds to or is reactive with anotherantigen on the surface of the target cell. Preferred additionalantibodies comprise at least one humanized, chimeric or human antibodyselected from the group consisting of an antibody reactive with CD4,CD5, CD8, CD14, CD15, CD16, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25,CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54,CD70, CD74, CD79a, CD80, CD95, CD126, CD133, CD138, CD154, CEACAM5,CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2, carbonic anhydrase IX, PAM4antigen, MUC1, MUC2, MUC3, MUC4, MUC5ac, Ia, MIF, HM1.24, HLA-DR,tenascin, Flt-3, VEGFR, PlGF, ILGF, IL-6, IL-25, tenascin, TRAIL-R1,TRAIL-R2, complement factor C5, oncogene product, or a combinationthereof. Various antibodies of use, such as anti-CD19, anti-CD20, andanti-CD22 antibodies, are known to those of skill in the art. See, forexample, Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al.,Cancer Immunol. Immunother. 32:364 (1991); Longo, Curr. Opin. Oncol.8:353 (1996), U.S. Pat. Nos. 5,798,554; 6,187,287; 6,306,393; 6,676,924;7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785; 7,238,786;7,282,567; 7,300,655; 7,312,318; 7,501,498; 7,612,180; 7,670,804; andU.S. Patent Application Publ. Nos. 20080131363; 20070172920;20060193865; and 20080138333, the Examples section of each incorporatedherein by reference.

The therapy can be further supplemented with the administration, eitherconcurrently or sequentially, of at least one therapeutic agent. Forexample, “CVB” (1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and150-200 mg/m² carmustine) is a regimen used to treat non-Hodgkin'slymphoma. Patti et al., Eur. J. Haematol. 51: 18 (1993). Other suitablecombination chemotherapeutic regimens are well-known to those of skillin the art. See, for example, Freedman et al., “Non-Hodgkin'sLymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.(eds.), pages 2028-2068 (Lea & Febiger 1993). As an illustration, firstgeneration chemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide,doxorubicin, vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate, bendamustine, and bryostatin-1.

The subject anti-IGF-1R complexes can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby theanti-IGF-1R complex is combined in a mixture with a pharmaceuticallysuitable excipient. Sterile phosphate-buffered saline is one example ofa pharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The subject anti-IGF-1R complexes can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Preferably, the complex is infused over a period of less than about 4hours, and more preferably, over a period of less than about 3 hours.For example, the first 25-50 mg could be infused within 30 minutes,preferably even 15 min, and the remainder infused over the next 2-3 hrs.Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the anti-IGF-1R complexes. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the anti-IGF-1R complexes. For example, biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of releasefrom such a matrix depends upon the molecular weight of the anti-IGF-1Rcomplex, the amount of complex within the matrix, and the size ofdispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The anti-IGF-1R complex may also be administered to a mammalsubcutaneously or even by other parenteral routes. Moreover, theadministration may be by continuous infusion or by single or multipleboluses. Preferably, the complex is infused over a period of less thanabout 4 hours, and more preferably, over a period of less than about 3hours.

More generally, the dosage of an administered anti-IGF-1R complex forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. It may be desirable to provide the recipient with a dosage ofanti-IGF-1R complex that is in the range of from about 1 mg/kg to 25mg/kg as a single intravenous infusion, although a lower or higherdosage also may be administered as circumstances dictate. A dosage of1-20 mg/kg for a 70 kg patient, for example, is 70-1,400 mg, or 41-824mg/m² for a 1.7-m patient. The dosage may be repeated as needed, forexample, once per week for 4-10 weeks, once per week for 8 weeks, oronce per week for 4 weeks. It may also be given less frequently, such asevery other week for several months, or monthly or quarterly for manymonths, as needed in a maintenance therapy.

Alternatively, an anti-IGF-1R complex may be administered as one dosageevery 2 or 3 weeks, repeated for a total of at least 3 dosages. Or, theconstruct may be administered twice per week for 4-6 weeks. If thedosage is lowered to approximately 200-300 mg/m² (340 mg per dosage fora 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may beadministered once or even twice weekly for 4 to 10 weeks. Alternatively,the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3months. It has been determined, however, that even higher doses, such as20 mg/kg once weekly or once every 2-3 weeks can be administered by slowi.v. infusion, for repeated dosing cycles. The dosing schedule canoptionally be repeated at other intervals and dosage may be giventhrough various parenteral routes, with appropriate adjustment of thedose and schedule.

In preferred embodiments, the anti-IGF-1R complexes are of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor). Cancers conducive to treatment methods of thepresent invention involves cells which express, over-express, orabnormally express IGF-1R.

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Exemplary autoimmune diseases include acute idiopathic thrombocytopenicpurpura, chronic immune thrombocytopenia, dermatomyositis, Sydenham'schorea, myasthenia gravis, systemic lupus erythematosus, lupusnephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid,pemphigus vulgaris, juvenile diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjögren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis and fibrosing alveolitis.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an antibody, antibody fragment, cytokine or constituentfusion protein of an anti-IGF-1R complex, such as a DNL construct.Fusion proteins may comprise an antibody or fragment or toxin attachedto, for example, an AD or DDD moiety.

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express the encoded protein(s) in a selected host cell,enhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG 1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine See Edelmanet al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969). In otherembodiments, an IgG1 sequence may be converted to an IgG4 sequence.

The skilled artisan will realize that methods of genetically engineeringexpression constructs and insertion into host cells to expressengineered proteins are well known in the art and a matter of routineexperimentation. Host cells and methods of expression of clonedantibodies or fragments have been described, for example, in U.S. Pat.Nos. 7,531,327 and 7,537,930, the Examples section of each incorporatedherein by reference.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain one or more anti-IGF-1R complexes as described herein. If thecomposition containing components for administration is not formulatedfor delivery via the alimentary canal, such as by oral delivery, adevice capable of delivering the kit components through some other routemay be included. One type of device, for applications such as parenteraldelivery, is a syringe that is used to inject the composition into thebody of a subject. Inhalation devices may also be used. In certainembodiments, a therapeutic agent may be provided in the form of aprefilled syringe or autoinjection pen containing a sterile, liquidformulation or lyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

General Techniques for Construction of Anti-IGF-1R Antibodies

The Vκ (variable light chain) and V_(H) (variable heavy chain) sequencesfor anti-IGF-1R antibodies may be obtained by a variety of molecularcloning procedures, such as RT-PCR, 5′-RACE, and cDNA library screening.The V genes of an anti-IGF-1R MAb from a cell that expresses a murineanti-IGF-1R MAb can be cloned by PCR amplification and sequenced. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V genesequences, a humanized anti-IGF-1R MAb can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine anti-IGF-1R MAb by general molecular cloningtechniques (Sambrook et al., Molecular Cloning, A laboratory manual,2^(nd) Ed (1989)). The Vκ sequence for the MAb may be amplified usingthe primers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extendedprimer set described by Leung et al. (BioTechniques, 15: 286 (1993)).The V_(H) sequences can be amplified using the primer pairVH1BACK/VH1FOR (Orlandi et al., 1989) or the primers annealing to theconstant region of murine IgG described by Leung et al. (Hybridoma,13:469 (1994)).

PCR reaction mixtures containing 10 μl of the first strand cDNA product,10 μl of 10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mMMgCl₂, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.),250 μM of each dNTP, 200 nM of the primers, and 5 units of Taq DNApolymerase (Perkin Elmer Cetus) can be subjected to 30 cycles of PCR.Each PCR cycle preferably consists of denaturation at 94° C. for 1 min,annealing at 50° C. for 1.5 min, and polymerization at 72° C. for 1.5min. Amplified Vκ and VH fragments can be purified on 2% agarose(BioRad, Richmond, Calif.). The humanized V genes can be constructed bya combination of long oligonucleotide template syntheses and PCRamplification as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the Vκ PCR products. PCR products for V_(H) can besubcloned into a similar staging vector, such as the pBluescript-basedVHpBS. Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al. (Proc. Natl.Acad. Sci., USA, 74: 5463 (1977)).

Expression cassettes containing the Vκ and V_(H) sequences, togetherwith the promoter and signal peptide sequences, can be excised fromVKpBR and VHpBS, respectively, by double restriction digestion asHindIII-BamHI fragments. The Vκ and V_(H) expression cassettes can beligated into appropriate expression vectors, such as pKh and pG1g,respectively (Leung et al., Hybridoma, 13:469 (1994)). The expressionvectors can be co-transfected into an appropriate cell, e.g., myelomaSp2/0-Ag14 (ATCC, VA), colonies selected for hygromycin resistance, andsupernatant fluids monitored for production of a chimeric, humanized orhuman anti-IGF-1R MAb by, for example, an ELISA assay. Alternatively,the Vκ and V_(H) expression cassettes can be assembled in the modifiedstaging vectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/BamHIfragments, respectively, and subcloned into a single expression vector,such as pdHL2, as described by Gillies et al. (J. Immunol. Methods125:191 (1989) and also shown in Losman et al., Cancer, 80:2660 (1997)).Another vector that is useful is the GS vector, as described in Barneset al., Cytotechnology 32:109-123 (2000). Other appropriate mammalianexpression systems are described in Werner et al., Arzneim.-Forsch./DrugRes. 48(11), Nr. 8, 870-880 (1998).

Co-transfection and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of VKpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992). Following transfection, cells may be grown in 96-wellmicrotiter plates in complete HSFM medium (Life Technologies, Inc.,Grand Island, N.Y.) at 37° C., 5% CO₂. The selection process can beinitiated after two days by the addition of hygromycin selection medium(Calbiochem, San Diego, Calif.) at a final concentration of 500 units/mlof hygromycin. Colonies typically emerge 2-3 weeks post-electroporation.The cultures can then be expanded for further analysis. Transfectomaclones that are positive for the secretion of chimeric, humanized orhuman heavy chain can be identified by ELISA assay.

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2μ membrane. The filtered medium is passed througha protein A column (1×3 cm) at a flow rate of 1 ml/min. The resin isthen washed with about 10 column volumes of PBS and protein A-boundantibody is eluted from the column with 0.1 M citrate buffer (pH 3.5)containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by absorbance at 280 nm and its concentration adjusted toabout 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is convenientlyadded to the sample as preservative.

EXAMPLES

The following examples are provided to illustrate, but not to limit, theclaims of the present invention.

Example 1. Generation and Initial Characterization of Anti-IGF-1RAntibodies: R1, cR1, and hR1

Three BALB/c mice were each immunized i.p. with 15 μg of recombinanthuman IGF-1R (R&D Systems, Catalog #391-GR), comprising a mixture ofboth processed and unprocessed extracellular domain of human IGF-1R, incomplete Freund's adjuvant. Additional immunizations in incompleteFreund's adjuvant were done 14, 21, and 28 days after the initialimmunization. Spleen cells from the immunized mice were fused withP3X63Ag8.653 cells to generate hybridomas according to standardprotocols. One clone (C-11) expressing anti-IGF-1R but not anti-IR(insulin receptor) activity was isolated and expanded in cultures toobtain the mouse antibody designated ML04R1 or R1, which was shown to bean IgG1/k with the ability to inhibit the binding of radioiodinatedIGF-1 to the IGF-1R expressing human breast cancer cell line MCF-7L (asubline of MCF-7) comparable to a commercially available mouseanti-IGF-1R monoclonal antibody (mAb) MAB391 (Table 6).

TABLE 6 Binding of ¹²⁵I-IGF-1 to MCF-7L in the presence of MAB391 or R1[Ab] MAB391^(a) R1 1000 ng/mL 38% 58% 100 ng/mL 54% 71% 10 ng/mL 95% 97%0 ng/mL 100%  100%  ^(a)R&D clone 33255.111

To obtain cR1, the mouse-human chimeric mAb of R1, the V_(H) and V_(K)genes of R1 were cloned by 5′-RACE. The authenticity of the cloned V_(H)and V_(K) genes was confirmed by N-terminal protein sequencing thatshowed an exact match of the first 15 N-terminal amino acids with thecorresponding amino acids deduced from DNA sequences (not shown). Thecloned V_(H) and V_(K) genes were inserted into the pdHL2 vector togenerate cR1pdHL2 (not shown), the expression vector for cR1.

cR1-producing clones were generated using SpE-26 (e.g., U.S. Pat. No.7,531,327), a variant of Sp2/0-Ag14 that shows improved growthproperties, as host cells. Briefly, approximately 30 μg of cR1pdHL2 waslinearized by digestion with SalI restriction endonuclease andtransfected into SpE-26 cells by electroporation. The transfectants wereselected with 0.075 μM methotrexate (MTX), and screened by ELISA forhuman Fc binding activities. The higher producing clones were furtherexpanded to pick the two best clones (709.2D2 and 710.2G2), from whichcR1 was produced in batch cultures, purified by Protein A, and eachconfirmed by ELISA to bind specifically to immobilized rhIGF-1R, but notto immobilized rhIR (not shown), with the same high affinity (K_(D)˜0.1nM) for immobilized rhIGF-1R (not shown). Surprisingly, cR1 appeared tohave a higher affinity than R1 for rhIGF-1R immobilized onto polystyrenebeads as shown by a competition assay in which the binding of R1 taggedwith a fluorescent probe was measured by flow cytometry in the presenceof varying concentrations of cR1 or R1 (not shown).

Successful humanization of cR1 to hR1 was achieved by grafting the CDRsonto the human framework regions of hMN-14 (U.S. Pat. Nos. 5,874,540 and6,676,924, the Examples section of each incorporated herein byreference) in which certain human framework residues were replaced withmurine counterparts of R1 at corresponding positions. Other selectedresidues were substituted for cloning purposes, resulting in the aminoacid sequences of hR1 V_(H) and hR1 V_(K) as shown in SEQ ID NO:94 andSEQ ID NO:95, respectively. Genes encoding hR1 V_(H) and hR1 Vk werethen synthesized and engineered into pdHL2 to obtain hR1pdHL2, theexpression vector for hR1. Subsequent efforts to secure the productionclone (711.3C11) for hR1 were similar to those describe above for cR1.Positive clones were selected for binding activity to rhIGF-1R. hR1displayed virtually the same binding affinity as cR1 for rhIGF-1Rimmobilized on polystyrene beads (not shown).

hR1 VH (SEQ ID NO: 94) QVQLQESGGGVVQPGRSLRLSCSASGFTFSDYYMYWVRQAPGKGLEWVAYITNYGGSTYYPDTVKGRFTISRDNAKNTLFLQMDSLRPEDTGVYFCARQSNYDYDGWFAYWGQGTPVTVSS  (SEQ ID NO: 95) hR1 VKDIQLTQSPSSLSASVGDRVTITCKASQEVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQ YSNYPLTFGQGTKVEIKR 

To determine whether cR1 can block the binding of IGF-1 or IGF-2 toIGF-1R, we used polystyrene beads immobilized with rhIGF-1R assurrogates of cells expressing IGF-1R and performed thebeads-competition assays as follows. Briefly, varying concentrations (0to 670 nM) of cR1, IGF-1, or IGF-2 were mixed with a constant amount of¹²⁵I-IGF-1 or ¹²⁵I-IGF-2. The rhIGF-1-coated beads were then added,incubated at room temperature for 1 h with gentle rocking, washed, andcounted for radioactivity. The results indicated that cR1 failed toblock the binding of either IGF-1 or IGF-2 to such immobilized rhIGF-1Runder these conditions (not shown). The results of a similar experimentalso indicated that binding of ¹²⁵I-IGF-1 to the bead-immobilized IGF-1Rwas effectively blocked by IGF-1 or MAB391, but not by hR1 or R1 (notshown). These findings suggest IGF-1 and MAB391 bind to the sameepitope, or have overlapping epitopes of IGF-1R, and hR1 targets adifferent region of IGF-1R from MAB391 or IGF-1. As the primary bindingsite of IGF-1R for IGF-1 was reported to be located in the cysteine-rich(CR) domain between amino acids (aa) 223 and 274, and the same region(aa 223-274) has been assigned as the epitope to αIR-3, which likeMAB391, competes for IGF-1 binding (Gustafson T A, Rutter W J. J BiolChem 1990; 265:18663-7), it appeared that MAB391 also binds to the sameregion or interacts with sites in close proximity.

Example 2. Epitope Mapping Studies of R1, cR1, and hR1

To further locate the region of IGF-1R to which hR1 binds, a panel ofcommercially available anti-IGF-1R mAbs that have their epitopes toIGF-1R mapped, were evaluated for their ability to cross-block eachother from binding to the IGF-1R-coated beads. The results showed thatbinding of R1 tagged with a fluorescent probe (PE) was not affected byMAB391 even at 100 μg/mL, and the binding of MAB391 tagged with PE wasonly partially inhibited (50 to 60%) by R1 at 100 μg/mL (not shown).Additional mapping studies indicate that the epitope of R1 is located inthe CR domain between aa 151 and 282 and can be further located to thefirst half of the CR domain between aa 151 and 222 (not shown).

Example 3. Additional Characterization of R1, cR1, and hR1

Whereas IGF-1 stimulates proliferation of MCF-7 cells grown inserum-free medium, achieving a maximal effect of 50% increase in viablecell counts at 100 ng/mL when compared to the untreated control at 48 h,hR1 does not (not shown). Thus hR1 is not agonistic upon binding toIGF-1R. Internalization of hR1 into MCF-7 was observed at 37° C. but notat 4° C. (not shown).

Example 4. Construction of Expression Vectors for hR1-IgG4(S228P)Variant

B13-24 cells containing an IgG4 gene are purchased from ATCC (ATCCNumber CRL-11397) and genomic DNA is isolated. Briefly, cells are washedwith PBS, resuspended in digestion buffer (100 mM NaCl, 10 mM Tris-HClpH8.0, 25 mM EDTA pH8.0, 0.5% SDS, 0.1 mg/ml proteinase K) and incubatedat 50° C. for 18 h. The sample is extracted with an equal volume ofphenol/chloroform/isoamylalcohol and precipitated with 7.5 MNH.sub.4Ac/100% EtOH. Genomic DNA is recovered by centrifugation anddissolved in TE buffer. Using genomic DNA as template, the IgG4 gene isamplified by PCR using the following primers.

Primer-SacII (SEQ ID NO: 96)CCGCGGTCACATGGCACCACCTCTCTTGCAGCTTCCACCAAGGGCCC  Primer-EagI:(SEQ ID NO: 97) CCGGCCGTCGCACTCATTTACCCAGAGACAGGG 

Amplified PCR product is cloned into a TOPO-TA sequencing vector(Invitrogen) and confirmed by DNA sequencing. The SacII-EagI fragmentcontaining the heavy chain constant region of IgG1 in hR1pdHL2 isreplaced with SacII-EagI of the TOPO-TA-IgG4 plasmid to produce thehR1-pdHL2-IgG4 (hR1pdHL2-γ4) vector.

IgG4-Proline Mutation

A Ser228Pro mutation is introduced in the hinge region of IgG4 to avoidformation of half-molecules. A mutated hinge region 56 bp fragment(PstI-StuI) is synthesized

(SEQ ID NO: 98) GAGTCCAAATATGGTCCCCCATGCCCACCGTGCCCAGGTAAGCCAACC CAGG;(SEQ ID NO: 99) CCTGGGTTGGCTTACCTGGGCACGGTGGGCATGGGGGACCATATTTGGACTCTGCAannealed and replaced with the PstI-StuI fragment of IgG4. Thisconstruction results in a final vector hR1pdHL2-γ4P.

Example 5. Generation of Multivalent hR1-Based Antibodies by DNL

The DNL technique may be used to make multivalent, hR1-based antibodiesin various formats that are either monospecific or bispecific. Forcertain preferred embodiments, Fab antibody fragments may be produced asfusion proteins containing either a DDD or AD sequence. Bispecificantibodies may be formed by combining a Fab-DDD fusion protein of afirst antibody with a Fab-AD fusion protein of a second antibody.Alternatively, an IgG-AD module may be produced as a fusion protein andcombined with a Fab-DDD module of the same or different specificity.Another alternative is a DDD-cytokine fusion, such as aDDD-interferon-α2b construct, combined with an anti-IGF-1R IgG-AD orFab-AD construct. Additional types of constructs may be made thatcombine the targeting capabilities of an antibody with the effectorfunction of any other protein or peptide.

Independent transgenic cell lines are developed for each DDD- orAD-fusion protein. Once produced, the modules can be purified if desiredor maintained in the cell culture supernatant fluid. Followingproduction, any Fab-AD or IgG-AD module can be combined with anyDDD-module, or any Fab-DDD module may be combined with any AD-module.DDD- or AD-modules may be produced synthetically such as linking anAD-sequence to polyethylene glycol or a DDD-sequence to anoligonucleotide. For different types of constructs, different AD or DDDsequences may be utilized.

DDD1: (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA  DDD2:(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA  AD1:(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA  AD2: (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC 

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See, Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD, Fab-AD, or IgG-ADexpression vectors, as described in detail below for Fab-DDD1 andFab-AD1. To generate the expression vector for Fab-DDD1, the codingsequences for the hinge, C_(H)2 and C_(H)3 domains of the heavy chainare replaced with a sequence encoding the first 4 residues of the hinge,a 14 residue Gly-Ser linker and DDD1 (the first 44 residues of humanRIIα). To generate the expression vector for Fab-AD1, the sequences forthe hinge, C_(H)2 and C_(H)3 domains of IgG are replaced with a sequenceencoding the first 4 residues of the hinge, a 15 residue Gly-Ser linkerand AD1 (a 17 residue synthetic AD called AKAP-IS, which was generatedusing bioinformatics and peptide array technology and shown to bind RIIαdimers with a very high affinity (0.4 nM). See Alto, et al. Proc. Natl.Acad. Sci., U.S.A. (2003), 100:4445-50).

To facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD1 orFab-AD1 expression vectors, two shuttle vectors were designed andconstructed as follows.

Preparation of C_(H)1

The C_(H)1 domain was amplified by PCR using the pdHL2 plasmid vector asa template. The left PCR primer consists of the upstream (5′) end of theC_(H)1 domain and a SacII restriction endonuclease site, which is 5′ ofthe C_(H)1 coding sequence. The right primer consists of the sequencecoding for the first 4 residues of the hinge followed by four glycinesand a serine (SEQ ID NO: 122), with the final two codons comprising aBam HI restriction site.

5′ of C_(H)1 Left Primer (SEQ ID NO: 100) 5′GAACCTCGCGGACAGTT AAG-3′C_(H)1 + G₄S-Bam Right  (″G₄S″ disclosed as SEQ ID NO: 122)(SEQ ID NO: 101) 5′GGATCCTCCGCCGCCGCAGCTCTTAGGTTTCTTGTCCACCTTGGTGTTGCTGG-3′

The 410 bp PCR amplimer was cloned into the pGemT PCR cloning vector(Promega, Inc.) and clones were screened for inserts in the T7 (5′)orientation.

Construction of (G₄S)₂DDD1 (“(G₄S)₂” disclosed as SEQ ID NO: 123)

A duplex oligonucleotide, designated (G₄S)₂DDD1 (“(G₄S)₂” disclosed asSEQ ID NO: 123), was synthesized by Sigma Genosys (Haverhill, UK) tocode for the amino acid sequence of DDD1 preceded by 11 residues of thelinker peptide, with the first two codons comprising a BamHI restrictionsite. A stop codon and an EagI restriction site are appended to the 3′end. The encoded polypeptide sequence is shown below.

(SEQ ID NO: 102) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

The two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom,that overlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGenosys) and combined to comprise the central 154 base pairs of the 174bp DDD 1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase.

RIIA1-44 top (SEQ ID NO: 103)5′GTGGCGGGTCTGGCGGAGGTGGCAGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCAGGGCTACACGGTGGAGGTGCTGCGAC AG-3′ RIIA1-44 bottom(SEQ ID NO: 104) 5′GCGCGAGCTTCTCTCAGGCGGGTGAAGTACTCCACTGCGAATTCGACGAGGTCAGGCGGCTGCTGTCGCAGCACCTCCACCGTGTAGCCC TG-3′

Following primer extension, the duplex was amplified by PCR using thefollowing primers:

G4S Bam-Left (″G4S″ disclosed as SEQ ID NO: 122) (SEQ ID NO: 105)5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3′ 1-44 stop Eag Right(SEQ ID NO: 106) 5′-CGGCCGTCAAGCGCGAGCTTCTCTCAGGCG-3′

This amplimer was cloned into pGemT and screened for inserts in the T7(5′) orientation.

Construction of (G₄S)₂-AD1 (“(G₄S)₂” Disclosed as SEQ ID NO: 123)

A duplex oligonucleotide, designated (G₄S)₂-AD1 (“(G₄S)₂” disclosed asSEQ ID NO: 123), was synthesized (Sigma Genosys) to code for the aminoacid sequence of AD1 preceded by 11 residues of the linker peptide withthe first two codons comprising a BamHI restriction site. A stop codonand an EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 107) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides, designated AKAP-IS Topand AKAP-IS Bottom, were synthesized.

AKAP-IS Top (SEQ ID NO: 108)5′GGATCCGGAGGTGGCGGGTCTGGCGGAGGTGGCAGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCTGACG  GCCG-3′ AKAP-IS Bottom(SEQ ID NO: 109) 5′CGGCCGTCAGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCTGCCACCTCCGCCAGACCCGCCACCTCCGG  ATCC-3′

The duplex was amplified by PCR using the following primers:

G4S Bam-Left (″G4S″ disclosed as SEQ ID NO: 122) (SEQ ID NO: 110)5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3′ AKAP-IS stop Eag Right(SEQ ID NO: 111) 5′-CGGCCGTCAGGCCTGCTGGATG-3′

This amplimer was cloned into the pGemT vector and screened for insertsin the T7 (5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from pGemT withBamHI and NotI restriction enzymes and then ligated into the same sitesin C_(H)1-pGemT to generate the shuttle vector C_(H)1-DDD1-pGemT.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from pGemTwith BamHI and NotI and then ligated into the same sites in CH1-pGemT togenerate the shuttle vector CH1-AD1-pGemT.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either C_(H)1-DDD1 or CH1-AD1 can beincorporated into any IgG-pdHL2 vector. The entire heavy chain constantdomain is replaced with one of the above constructs by removing theSacII/EagI restriction fragment (C_(H)1-C_(H)3) from pdHL2 and replacingit with the SacII/EagI fragment of C_(H)1-DDD1 or C_(H)1-AD1, which isexcised from the respective pGemT shuttle vector.

CH1-DDD2-Fab-hR1-pdHL2

C_(H)1-DDD2-Fab-hR1-pdHL2 is an expression vector for production ofC_(H)1-DDD2-Fab-hR1, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd via a 14amino acid residue Gly/Ser peptide linker.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide (GGGGSGGGCG, SEQ ID NO:112) and residues 1-13of DDD2, were made synthetically. The oligonucleotides were annealed andphosphorylated with T4 PNK, resulting in overhangs on the 5′ and 3′ endsthat are compatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

G4S-DDD2 top (″G4S″ disclosed as SEQ ID NO: 122) (SEQ ID NO: 113)5′GATCCGGAGGTGGCGGGTCTGGCGGAGGTTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3′ G4S-DDD2 bottom (″G45″disclosed as SEQ ID NO: 122) (SEQ ID NO: 114)5′GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCAACCTC CGCCAGACCCGCCACCTCCG-3′

The duplex DNA was ligated with the shuttle vector CH1-DDD1-pGemT, whichwas prepared by digestion with BamHI and PstI, to generate the shuttlevector CH1-DDD2-pGemT. A 507 bp fragment was excised from CH1-DDD2-pGemTwith SacII and EagI and ligated with the IgG expression vector hR1pdHL2,which was prepared by digestion with SacII and EagI. The finalexpression construct is C_(H)1-DDD2-Fab-hR1-pdHL2.

Generation of C_(H)1-AD2-Fab-h679-pdHL2

C_(H)1-AD2-Fab-h679-pdHL2 is an expression vector for the production ofC_(H1)-AD2-Fab-h679 and is useful as a template for the DNA sequenceencoding AD2. The expression vector is engineered as follows. Twooverlapping, complimentary oligonucleotides (AD2 Top and AD2 Bottom),which comprise the coding sequence for AD2 and part of the linkersequence, are made synthetically. The oligonucleotides are annealed andphosphorylated with T4 polynucleotide kinase, resulting in overhangs onthe 5′ and 3′ ends that are compatible for ligation with DNA digestedwith the restriction endonucleases BamHI and SpeI, respectively.

AD2 Top (SEQ ID NO: 115) 5′GATCCGGAGGTGGCGGGTCTGGCGGATGTGGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCGGCTGCTG AA-3′ AD2 Bottom(SEQ ID NO: 116) 5′TTCAGCAGCCGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCCACATCCGCCAGACCCGCCACCTCCG-3′

The duplex DNA is ligated into the shuttle vector C_(H)1-AD1-pGemT,which is prepared by digestion with BamHI and SpeI, to generate theshuttle vector CH1-AD2-pGemT. A 429 base pair fragment containing C_(H)1and AD2 coding sequences is excised from the shuttle vector with SacIIand EagI restriction enzymes and ligated into h679-pdHL2 vector that isprepared by digestion with those same enzymes, resulting inC_(H)1-AD2-Fab-h679-pdHL2.

Generation of C_(H)3-AD2-IgG-pdHL2 for Expressing C_(H3)-AD2-IgG

C_(H)3-AD2-IgG modules have an AD2 peptide fused to the carboxylterminus of the heavy chain of IgG via a 9 amino acid residue peptidelinker. The DNA coding sequences for the linker peptide (GSGGGGSGG, SEQID NO:117) followed by the AD2 peptide are coupled to the 3′ end of theC_(H)3 (heavy chain constant domain 3) coding sequence by standardrecombinant DNA methodologies, resulting in a contiguous open readingframe. When the heavy chain-AD2 polypeptide is co-expressed with a lightchain polypeptide, an IgG molecule is formed possessing two AD2peptides, which can therefore bind two Fab-DDD2 dimers. TheC_(H)3-AD2-IgG module can be combined with any C_(H)1-DDD2-Fab module togenerate a wide variety of hexavalent structures composed of an Fcfragment and six Fab fragments. If the C_(H)3-AD2-IgG module and theC_(H)1-DDD2-Fab module are derived from the same parental monoclonalantibody (MAb) the resulting complex is monospecific with 6 binding armsto the same antigen. If the modules are instead derived from twodifferent MAbs then the resulting complexes are bispecific, with twobinding arms for the specificity of the C_(H)3-AD2-IgG module and 4binding arms for the specificity of the C_(H)1-DDD2-Fab module.

A plasmid shuttle vector was produced to facilitate the conversion ofany IgG-pdHL2 vector into a C_(H)3-AD2-IgG-pdHL2 vector. The gene forthe Fc (C_(H)2 and C_(H)3 domains) was amplified using the pdHL2 vectoras a template and the oligonucleotides Fc BglII Left and Fc Bam-EcoRIRight as primers.

Fc BglII Left (SEQ ID NO: 118) 5′-AGATCTGGCGCACCTGAACTCCTG-3′Fc Bam-EcoRI Right (SEQ ID NO: 119)5′-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3′

The amplimer was cloned in the pGemT PCR cloning vector. The Fc insertfragment was excised from pGemT with XbaI and BamHI restriction enzymesand ligated with AD2-pdHL2 vector that was prepared by digestion ofC_(H)1-AD2-Fab-h679-pdHL2 with XbaI and BamHI, to generate the shuttlevector Fc-AD2-pdHL2.

To convert any IgG-pdHL2 expression vector to a C_(H)3-AD2-IgG-pdHL2expression vector, an 861 bp BsrGI/NdeI restriction fragment is excisedfrom the former and replaced with a 952 bp BsrGI/NdeI restrictionfragment excised from the Fc-AD2-pdHL2 vector. BsrGI cuts in the C_(H)3domain and NdeI cuts downstream (3′) of the expression cassette.

Example 6. Generation of Hex-hR1

The DNL method was used to create Hex-hR1, a monospecific anti-IGF-1Rwith one Fc and six Fabs, by combining C_(H3)-AD2-IgG-hR1 withC_(H)1-DDD2-Fab-hR1. Hex-hR1 was made in four steps.

Step 1, Combination:

C_(H)1-DDD2-Fab-hR1 was mixed with C_(H)3-AD2-IgG-hR1 in phosphatebuffered saline, pH 7.4 (PBS) with 1 mM EDTA, at a molar ratio of 4.2such that there are two C_(H)1-DDD2-Fab-hR1 for each AD2 onC_(H)3-AD2-IgG-hR1, allowing some excess of C_(H)1-DDD2-Fab-hR1 toensure that the coupling reaction was complete.

Step 2, Mild Reduction:

Reduced glutathione (GSH) was added to a final concentration of 1 mM andthe solution held at room temperature (16-25° C.) for 1 to 24 hours.

Step 3, Mild Oxidation:

Following reduction, oxidized glutathione (GSSH) was added directly tothe reaction mixture to a final concentration of 2 mM and the solutionwas held at room temperature for 1 to 24 hours.

Step 4, Isolation of the DNL Product:

Following oxidation, the reaction mixture was loaded directly onto aProtein-A affinity chromatography column. The column was washed with PBSand the Hex-hR1 eluted with 0.1 M Glycine, pH 2.5. The unreactedC_(H)1-DDD2-Fab-hR1 was removed from the desired product in the unboundfraction. Other hexavalent DNL constructs can be prepared similarly bymixing a selected pair of C_(H)3-AD2-IgG and C_(H)1-DDD2-Fab.

A list of such DNL constructs and structural controls related to thepresent invention is provided in Table 7. Each of these constructs wasshown to retain the binding activities of the constitutive antibodies.

TABLE 7 hR1-containing DNL constructs and structural controls DNL IgG-Fab- Valency code AD2 DDD2 2 4 6 Hex- hR1 hR1 — — IGF-1R hR1 Hex- hRS7hRS7 — — EGP-1 hRS7 Hex- hPAM4 hPAM4 — — mucin hPAM4 Hex- hMN- hMN14 — —CEACAM5 hMN-14 14 Hex- hLL1 hLL1 — — CD74 hLL1 Hex- hL243 hL243 — —HLA-DR hL243 1R-E1 hR1 hRS7 IGF-1R EGP-1 — 1R-14 hR1 hMN- IGF-1R CEACAM5— 14 1R-15 hR1 hMN- IGF-1R CEACAM6 — 15 1R-31 hR1 hAFP IGF-1R AFP —1R-74 hR1 hLL1 IGF-1R CD74 — 1R-C2 hR1 hL243 IGF-1R HLA-DR — 1R-M1 hR1hPAM4 IGF-1R mucin E1-1R hRS7 hR1 EGP-1 IGF-1R — M1-1R hPAM4 hR1 mucinIGF-1R — 14-1R hMN- hR1 CEACAM5 IGF-1R — 14 74-1R hLL1 hR1 CD74 IGF-1R —C2-1R hL243 hR1 HLA-DR IGF-1R — 22-20 hLL2 hA20 CD22 CD20 —

Example 7. Production of AD- and DDD-Linked Fab and IgG Fusion Proteinsfrom Multiple Antibodies

The following IgG or Fab fusion proteins were constructed andincorporated into DNL constructs, retaining the antigen-bindingcharacteristics of the parent antibodies.

TABLE 8 Fusion proteins comprising IgG or Fab Moieties Fusion ProteinBinding Specificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSGC-(AD)₂-Fab-h679 HSG C-AD2-Fab-h734 Indium-DTPA C-AD2-Fab-hA20 CD20C-AD2-Fab-hA20L CD20 C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22N-AD2-Fab-hLL2 CD22 C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1RC-AD2-IgG-hRS7 EGP-1 C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74C-DDD1-Fab-hMN-14 CEACAM5 C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSGC-DDD2-Fab-hA19 CD19 C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFPC-DDD2-Fab-hL243 HLA-DR C-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22C-DDD2-Fab-hMN-3 CEACAM6 C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUCC-DDD2-Fab-hR1 IGF-1R C-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

Example 8. IGF-1R Expression in Cancer Cell Lines

Zenon-labeled various parental antibodies as well as multivalentantibodies derived from these antibodies were used to assess theexpression levels of cognate antigens in several cancer cell lines byflow cytometry performed on Guava instrument. Expression of IGF-1R wasconfirmed by the binding of hR1 to MCF-7 (breast cancer), CaPan1(pancreatic cancer), and DU-145 (prostate cancer) (not shown). The dualexpression of IGF-1R and AFP in HepG2 (liver cancer) was also shown bythe binding of humanized anti-AFP IgG and TF18 (made by combiningC_(H)1-DDD2-Fab-hAFP with C_(H1)-AD2-Fab-h679 to contain two Fabfragments of hAFP), as well as by the enhanced binding of hRl-IgG-AD2(the dimer of C_(H)3-AD2-IgG-hR1) and 1R-31, suggesting a higheraffinity of these multivalent DNL constructs (not shown). The expressionof CEACAM6 in Hep G2 was evidenced by the enhanced binding of 1R-15.Additional studies performed with MCF-7, DU-145, and ME-180 (cervicalcancer) are summarized in Table 9, which shows that the multivalent DNLconstructs exhibit enhanced binding to target cell lines compared totheir parental antibodies.

TABLE 9 Flow cytometry data obtained from FACScan MCF-7 DU145 ME-180Antibody MFI % Positive Antibody MFI % Positive Antibody MFI % Positive/ / — 2.4 1.96 — 1.86 2 human IgG 1.84 2.34 human IgG 2.21 1.44 humanIgG 1.8 1.37 22-20 2.11 3.1 DNL1 2.74 7.88 DNL1 1.98 12.6 hR1 9.93 89.15hR1 5.33 30.39 hR1 3.65 10.74 hRS7 21.42 99.15 hRS7 10.58 82.82 hRS735.54 99.96 Hex-hR1 14.08 98.58 Hex-hR1 7.83 72.56 Hex-hR1 6.36 33.02Hex-hRS7 35.73 99.86 Hex-hRS7 17.03 93.74 Hex-hRS7 59.58 99.95 1R-E147.85 99.92 1R-E1 22.10 99.53 1R-E1 76.29 99.94 E1-1R 109.19 98.77 E1-1R53.96 99.9 E1-1R 254.8 99.89

Example 9. Neutralizing Activity of Hex-hR1 and 1R-E1

The following experiments were performed to determine the effect ofHex-hR1 or 1R-E1 on neutralizing the growth stimulating activity ofIGF-1 in DU-145 and ME-180, both of which express IGF-1R and EGP-1.Target cells were seeded at 2000/well onto 96-well plates and grownovernight in complete medium. Cells were washed twice with serum freemedium and exposed to a selected multivalent antibody at 0.8, 4, 20, and100 μg/mL in serum free medium for 2 h, followed by the addition ofIGF-1 to a final concentration of 10 ng/ml. Cells were incubated for 72hours and then subjected to MTS assay. Under these conditions, Hex-hR1suppressed the proliferation of DU-145 and ME-180 in a dose-dependentmanner (not shown) with statistical significance. Similar results wereobtained with 1R-E1 in ME-180 (not shown).

Example 10. Downregulation of IGF-1R

One major mechanism of anti-tumor actions induced by an anti-IGF-1Rantibody, despite its being an agonist or antagonist, is to downregulateIGF-1R via endocytosis leading to subsequent degradation in endosomalvesicles. Efficient downregulation of IGF-1R in MCF-7 or HT-29(colorectal cancer) was clearly demonstrated with hR1 at 100 nM as wellas the two commercially available anti-IGF-1R antibodies (MAB391 and24-60) serving as positive controls, but not with the anti-CD22antibody, hLL2 (epratuzumab), which serves as a negative control (notshown). Further studies revealed that Hex-hR1 and 1R-E1 were capable ofsubstantially reducing the level of IGF-1R at a concentration as low as0.1 nM in MCF-7, DU-145, and LnCap (androgen-dependent prostate cancer)(not shown).

Example 11. Anti-Tumor Effects of Multivalent Anti-IGF-1R Complexes areEnhanced in Renal Cell Carcinoma and Synergistic with an mTOR Inhibitor

Among kidney cancer types, approximately 90% are renal cell carcinomas(RCC). Advanced or metastatic RCC, which presents in about one third ofthe patients, has a poor prognosis, because it is resistant toconventional chemotherapy or radiotherapy. Treatments with humaninterferon-α2b (IFN-α2b) alone or in combination with mTOR inhibitorssuch as rapamycin have led to only modest improvements in outcome. Oneobservation made with mTOR inhibitors is that cancer cells can overcomethe effects of the inhibitor by activating the insulin-like growthfactor-I (IGF-I) signaling pathways. Clinically, there is an associationof IGF-I receptor (IGF-IR) expression in RCC and poor long-term patientsurvival, particularly among patients with high-grade tumors.

A humanized anti-IGF-IR monoclonal antibody, hR1, binds to multipletumor types, including RCC, resulting in effective down-regulation ofIGF-IR and moderate inhibition of cell proliferation in vitro. Toenhance the anti-tumor activity of hR1, we generated the DOCK-AND-LOCK™(DNL™) complex 1R-2b, comprising a conjugate of hR1 IgG with two dimersof interferon-α2b, and Hex-hR1, comprising 6 Fab fragments of hR1tethered onto a common Fc. To make Hex-R1, a dimerization and dockingdomain (DDD) was fused to hR1 Fab to product a self-associating dimericFab-DDD2. An anchor domain (AD) was fused to the two CH3 domains of hR1IgG to produce a CH3-AD2-IgG molecule with two AD peptides. Finalassembly was readily obtained by mixing the Fab-DDD2 with theCH3-AD2-IgG under mild redox conditions, to produce a DNL™ complexcomprising four Fab moieties attached to an IgG moiety. To produce1R-2b, DDD2 was fused to human IFN-α2b and the DDD2-IFN-α2b moiety wasmixed with a CH3-AD2-IgG molecule under mild redox conditions.

There was no loss in cell binding for either 1R-2b or Hex-hR1 whencompared to parental hR1 as determined by flow cytometry (not shown).The IFN-α specific activity was measured at 3750 U/pmole for 1R-2bversus 180 U/pmole and 3255 U/pmole for two different forms ofpeginterferon alpha-2 (60 and 31 kDa), respectively with a luciferasereporter gene fused to a promoter containing the interferon-stimulatedresponse element (iLite kit).

An in vitro cytotoxicity assay with 1R-2b demonstrated growth inhibitionof two different RCC cell lines, 786-0 and ACHN, with EC50-values of0.049 and 0.062 pmole/mL, respectively (FIG. 1). Hex-hR1 induced thedown-regulation of IGF-IR at 10-fold lower concentrations compared tothe parental hR1 IgG (FIG. 1). In soft-agar growth assays, all threeagents (hR1, Hex-hR1 and 1R-2b) significantly inhibited colony formationof 786-0 and ACHN (P<0.038 and P<0.0022, respectively) (FIG. 1). Table10 summarizes the data on growth inhibition by different forms ofanti-IGF-1R for Caki-2 cells, ACHN cells and 786-0 cells.

TABLE 10 Maximum growth inhibition by Anti-IGF-1R under serum-freeconditions. Data were obtained from the experiments shown in FIG. 1.Concentration at maximum inhibition in parentheses. Caki-2 Cells ACHNCells 786-O Cells Antibody (8 nM) (200 nM) (8 nM) hR1  33 ± 0.6%  3 ±4.4% 26 ± 5.4% Hex-hR1 *43 ± 0.3% *48 ± 1.9%  35 ± 4.8% MAB 391 *50 ±4.7% *48 ± 13.2% 34 ± 2.0% *Significantly different from hR1 (P < 0.001)

The activity of the 1R-2b DNL™ complex, comprising four copies ofIFN-α2b attached to hR1 IgG, was examined. Based on a luciferasereporter gene assay (iLite kit), 1R-2b yielded a specific activity of15×10⁶ U/mg or 3750 U/pmole versus 180 and 3255 U/pmole for twodifferent pegylated-IFN molecules (FIG. 2A). In growth inhibition assaysof 786-0 (FIG. 2B) and ACHN (FIG. 2C), 1R-2b had EC50 values of 49 and62 pM, respectively. Confirmation of units of activity was furtherdemonstrated in IFN-mediated phosphorylation of STAT1. Cells were platedin 6-well plates overnight. On the following day IFN, either in the formof rhIFN-a2a or 1R-2b, was added at 100, 10 and 1 U/ml of IFN. After 30min, cell lysates were prepared and resolved by SDS-PAGE, transferred tonitrocellulose membranes and probed with antibodies to phospho-STAT1.ACHN cells showed similar levels of pSTAT1 for 1R-2b and rhIFN-α2a ateach of the three different concentrations added to the plates (notshown). The ratios of pSTAT1 relative to untreated (β-actin control) forwere respectively 13.1 (rhIFN-α at 100 U/ml IFN), 2.3 (rhIFN-α at 10U/ml IFN), 1.3 (rhIFN-α at 1 U/ml IFN), 13.7 (1R-2b at 100 U/ml IFN),2.4 (1R-2b at 10 U/ml IFN), 1.1 (1R-2b at 1 U/ml IFN) and 1.0 (control).1R-2b and rhIFN-α2a had similar effects in 786-O cells (not shown).

The effect of hR1 constructs on growth inhibition underanchorage-independent conditions was examined (FIG. 3). A 1% base agarwas mixed 1:1 with 2× growth media (10% FBS final concentration) andadded to wells of a 24-well plate. Cells in 2× growth media were mixed1:1 with 0.7% agarose and added (1250 cells per well) to the base agar.Cells were fed by weekly replacement of growth media on the top of theagarose layer. Treated wells contained the test articles in theagarose/cell layer at the beginning and in subsequent feedings. Oncecolonies were clearly visible by microscopy in untreated control wells,the medium was removed and the colonies stained with crystal violet.Colonies were counted under a microscope and the average number wasdetermined from five different fields of view within the well. Each ofthe hR1 constructs induced significant growth inhibition of ACHN cellsunder anchorage-independent conditions (FIG. 3). Both Hex-hR1 and 1R-2bshowed significantly greater growth inhibition compared to unconjugatedhR1, with the greatest effect shown by the IFN-α2b DNL™ construct (FIG.3). hR1 IgG and 1R-2b, but not Hex-hR1, significantly inhibitedanchorage-independent growth of 786-0 cells, with the greatest effectagain observed with the IFN-α2b DNL™ construct (FIG. 3).

When combined with temsirolimus, an mTOR inhibitor, in vitrocytotoxicity assays demonstrated a synergistic interaction with hR1,Hex-hR1, and 1R-2b. This synergy occurred at concentrations as low as 10nM for hR1, 1 nM for Hex-hR1, and 2.6 nM for 1R-2b (FIG. 4). ACHN cellswere harvested, washed in PBS several times to remove FBS, and plated in96-wells plates overnight in SFM. On the following day, various doses (1mM to 0.06 nM) of the mTOR inhibitor temsirolimus was added to theplates with and without hR1 or Hex-hR1 (100, 10, and 1 nM constantamounts) or 1R-2b (26, 2.6, or 0.26 nM; NOTE: 26 nM 1R-2b 100,000Units/mL of IFN). IGF-1 was added at 100 ng/mL. Plates were incubatedfor 96-h before MTS substrate was added to all the wells and the platesread at 492 nm. Data was graphed as Percent Growth Inhibition vs.[temsirolimus]. IC50-values for temsirolimus were determined for eachcondition and Combinatorial Index (CI) was calculated based on changesin these values when co-incubated with hR1, Hex-hR1, or 1R-2b (CI<1 forsynergy). FIG. 4A shows the effect of the combination of temsirolimuswith hR1 (CI=0.64). The IC₅₀ values for temsirolimus concentrationneeded to mediate 50% inhibition of cell growth were 7.76 nM for Temalone (R² 0.94); 1.45 nM with 100 nM hR1 (R² 0.88); 0.56 nM with 10 nMhR1 (R² 0.84); and 2.86 nM with 1 nM hR1 (R² 0.93). FIG. 4B shows theeffect of the combination of temsirolimus with Hex-hR1 (CI=0.43). TheIC₅₀ values were 7.76 nM for Tem alone (R² 0.94); 3.15 nM with 1 nMHex-hR1 (R² 0.63); 0.06 nM with 10 nM Hex-hR1 (R² 0.66); and <0.06 nMwith 100 nM HexhR1 (R² 0.63). FIG. 4C shows the effect of thecombination of temsirolimus with 1R-2b (CI=0.02). The IC₅₀ values were7.76 nM for Tem alone (R² 0.94); <0.06 nM with 26 nM 1R-2b (R² 0.32);<0.06 nM with 2.6 nM 1R-2b (R² 0.34); and 12.7 nM with 0.26 nM 1R-2b (R²0.81).

In conclusion, two novel anti-IGF-1R DNL™ complexes were created for thetreatment of RCC. Both Hex-hR1 and 1R-2b retained anti-IGF-1R bindingcapacity to target cells, similar to that of the parental hR1. Hex-hR1mediated complete receptor down-regulation at concentrations as low as 1nM versus 10 nM for hR1. 1R-2b demonstrated signaling activity similarto rhIFN-α2a in two different RCC cell lines. Both Hex-hR1 and 1R-2binhibit the growth of RCC tumor lines in vitro and underanchorage-independent growth in soft-agar. Synergistic interactions wereobserved with these anti-IGF-1R molecules when combined with the mTORinhibitor, temsirolimus. These data suggest that while each agent showsactivity when used alone, a greater benefit might be achieved whencombined with an mTOR inhibitor.

Example 12. Bispecific, Hexavalent Antibodies Targeting IGF-1R andTrop-2 or CEACAM6 Inhibit Anchorage-Independent Growth and Invasion ofBreast and Pancreatic Cancer Cell Lines

Combination therapy using two distinct monoclonal antibodies to achieveimproved efficacy without increased toxicity is being pursued in variouspreclinical and clinical studies. Preferably, this may be accomplishedwith a single bispecific antibody to avoid the need for administeringtwo antibodies sequentially, which is time consuming, expensive andinconvenient. Based on the elevated expression of the type Iinsulin-like growth factor receptor (IGF-1R), the trophoblastcell-surface marker (Trop-2), and the carcinoembryonic antigen relatedcell adhesion molecule 6 (CEACAM6) in diverse epithelial cancer celllines, we explored the potential of 1R-(E1)-(E1) and 1R-(15)-(15), twonovel bispecific HexAbs targeting IGF-1R/Trop-2 and IGF-1R/CEACAM6,respectively, for treating breast and pancreatic cancers.

1R-(E1)-(E1), comprising the IgG of hR1 (humanized anti-IGF-1R) and fourFabs of hRS7 (humanized anti-Trop-2) was generated as a DNL™ complex byreacting the IgG-AD2 module of hR1 with the Fab-DDD2 module of hRS7under mild redox conditions, followed by purification on Protein A.1R-(15)-(15) was generated in a similar fashion using the Fab-DDD2module of hMN-15 (humanized anti-CEACAM6).

The in vitro effects of 1R-(E1)-(E1) and 1R-(15)-(15) on threetriple-negative breast cancer lines of varying invasive activities(MCF-7, low; MDA-MB-468, moderate; MDA-MB-231, high) and two pancreaticcancer lines (Capan-1 and BxPC-3) were determined, which included cellbinding by flow cytometry, anchorage-independent growth by soft agarassay, and invasiveness by BD matrigel chambers. Statistical differences(P values) between two populations were determined by Student's t-test.

All five cell lines were found to express IGF-1R, Trop-2, and CEACAM6 ofsufficient levels, which are higher in MCF-7, MDA-MB-468, and BxPC-3,respectively (not shown). When tested at 100 μg/mL, 1R-(E1)-(E1) reducedthe invasion of MDA-MB-468 to less than 10% of the untreated control(not shown), whereas under the same conditions, MDA-MB-231 appeared tobe resistant and the parental antibodies showed no effect (not shown).1R-(15)-(15) at 100 μg/mL potently reduced the invasion of Capan-1, buthad little effect on MDA-MB-468 (not shown). The ability of 1R-(E1)-(E1)to inhibit anchorage-independent growth was demonstrated at 200 nM inMDA-MB-231 with statistically significant difference (P<0.041) whencompared with samples treated with parental antibodies at the sameconcentrations (not shown). Cells treated with 1R-(E1)-(E1) produced fewand much smaller colonies, the largest size of which was less than 1/10of the untreated cells (not shown). The parental hR1 alone, but nothRS7, had some effect on inhibiting the growth of MDA-MB-231 in softagar, presumably resulting from the downregulation of IGF-1R (notshown). These results evidence the potential of bispecific HexAbs fortargeted therapy of solid cancers.

The skilled artisan will realize that the disclosed methods andcompositions are not limited to the specific anti-IGF-1R antibodies, DNLconstructs and/or mTOR inhibitors described above, but rather maycomprise other known anti-IGF-1R antibodies or antigen-binding fragmentsthereof, other known mTOR inhibitors and alternative constructs.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and used without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations may be applied to the COMPOSITIONS and METHODS andin the steps or in the sequence of steps of the METHODS described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A complex comprising: (a) an anti-IGF-1R(insulin-like growth factor type I receptor) IgG antibody attached to ananchor domain (AD) moiety from an A-kinase anchoring protein (AKAP),wherein the anti-IGF-1R antibody comprises the heavy chain CDRsequences: CDR1 having the amino acid sequence DYYMY (SEQ ID NO:85),CDR2 having the amino acid sequence YITNYGGSTYYPDTVKG (SEQ ID NO:86) andCDR3 having the amino acid sequence QSNYDYDGWFAY (SEQ ID NO:87) and thelight chain CDR sequences: CDR1 having the amino acid sequenceKASQEVGTAVA (SEQ ID NO:88), CDR2 having the amino acid sequence WASTRHT(SEQ ID NO:89) and CDR3 having the amino acid sequence QQYSNYPLT (SEQ IDNO:90); and (b) two copies of a fusion protein, each fusion proteincomprising an effector attached to one copy of a dimerization anddocking domain (DDD) moiety from a human protein kinase A (PKA)regulatory subunit RIα, RIβ, RIIα or RIIβ; wherein the effector is acytokine or a F(ab′)₂, Fab, Fab′ or scFv from a second antibody.
 2. Thecomplex of claim 1, wherein the second antibody binds to atumor-associated antigen selected from the group consisting of carbonicanhydrase IX, CSAp (colon-specific antigen-p), CD1 (cluster ofdifferentiation 1), CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16,CD18, CD19, CD20, IGF-1R, CD21, CD22, CD23, CD25, CD29, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64,CD66a, CD66b, CD66c, CD66d, CD66e, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5 (carcinoembryonicantigen-related cell adhesion molecule 5), CEACAM6, ED-B (extradomain B)of fibronectin, Factor H, FHL-1 (four and a half LIM domains protein 1),Flt-3 (Fms-like tyrosine kinase 3), folate receptor, GRO-β (growthregulatory oncogene beta), HMGB-1 (high mobility group protein B1),hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1(ILGF-1), IFN-γ (interferon-gamma), IFN-α, IFN-β, IL-2 (interleukin-2),IL-4R (interleukin-4 receptor), IL-6R, IL-13R, IL-15R, IL-17R, IL-18R,IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10 (interferongamma-induced protein 10), MAGE (melanoma antigen), mCRP (modifiedC-reactive protein), MCP-1 (monocyte chemoattractant protein-1), MIP-1A(macrophage inflammatory protein-1A), MIP-1B, MIF (macrophage migrationinhibitory factor), MUC1 (mucin 1), MUC2, MUC3, MUC4, MUC5ac, NCA-95(normal glycoprotein crossreacting with CEA-95), NCA-90, PSMA(prostate-specific membrane antigen), EGP-1 (epithelial/carcinomaantigen-1), EGP-2, AFP (alpha fetoprotein), HLA-DR (human leukocyteantigen-DR), tenascin, Le(y) (Lewis antigen y), RANTES(regulated onactivation, normal T cell expressed and secreted), Tn (Thomsen-nouvelle)antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α(tumor necrosis factor-alpha), TRAIL (TNF-related apoptosis-inducingligand) receptor R1, TRAIL receptor R2, VEGFR (vascular endothelialgrowth factor receptor), EGFR (epidermal growth factor receptor), P1GF(placental growth factor), complement factor C3, complement factor C3a,complement factor C3b, complement factor C5a, complement factor C5, andan oncogene product.
 3. The complex of claim 1, wherein the cytokine isselected from the group consisting of human growth hormone, N-methionylhuman growth hormone, bovine growth hormone, parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, thrombopoietin (TPO), NGF-β,platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I,insulin-like growth factor-II, erythropoietin (EPO), osteoinductivefactors, an interferon, interferon-α, interferon-β, interferon-γ,macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-25, kit-ligand, angiostatin, thrombospondin, andendostatin.
 4. The complex of claim 1, wherein the cytokine isinterferon-α2b.
 5. The complex of claim 1, wherein the anti-IGF-1Rantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO:94 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:95.
 6. The complex ofclaim 1, wherein the anti-IGF-1R antibody is a chimeric antibody or ahumanized antibody.
 7. The complex of claim 1, wherein the antibodycomprises an Fc region capable of inducing effector function, whereinthe complex is effective in treating a cancer that expresses humanIGF-1R.
 8. The complex of claim 7, wherein the cancer is selected fromthe group consisting of Wilms' tumor, Ewing sarcoma, a neuroendocrinetumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breastcancer, colon cancer, rectal cancer, prostate cancer, liver cancer,renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervicalcancer, endometrial cancer, esophageal cancer, gastric cancer, head andneck cancer, medullary thyroid carcinoma, ovarian cancer, glioma,lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acutemyelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenousleukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and urinarybladder cancer.
 9. The complex of claim 7, wherein the cancer is renalcell carcinoma, breast cancer or pancreatic cancer.
 10. The complex ofclaim 1, further comprising a therapeutic agent selected from the groupconsisting of a radionuclide, an immunomodulator, an anti-angiogenicagent, a cytokine, a chemokine, a growth factor, a hormone, a drug, aprodrug, an enzyme, a pro-apoptotic agent, a photoactive therapeuticagent, a cytotoxic agent, a chemotherapeutic agent and a toxin, whereinthe therapeutic agent is conjugated to the complex.
 11. The complex ofclaim 10, wherein the drug is selected from the group consisting of5-fluorouracil, azaribine, anastrozole, anthracyclines, bleomycin,bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin,carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, cladribine, camptothecans, cyclophosphamide, cytarabine,dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-d0), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vinorelbine, vinblastine, vincristine andvinca alkaloids.
 12. The complex of claim 10, wherein the toxin isselected from the group consisting of ricin, abrin, saporin,ribonuclease (RNase), ranpirnase DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin.
 13. The complex of claim 10,wherein the radionuclide is selected from the group consisting of ₁₁₁In,¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰ Y, ¹²⁵I, ¹³¹I, ³²P, ³³P,⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd,¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and 211Pb.
 14. A compositioncomprising the complex of claim 1 and a buffer.
 15. A complexcomprising: (a) an anti-IGF-1R antigen-binding antibody fragmentattached to an anchor domain (AD) moiety from an A-kinase anchoringprotein (AKAP), wherein the anti-IGF-1R antigen-binding antibodyfragment comprises the heavy chain CDR sequences: CDR1 having the aminoacid sequence DYYMY (SEQ ID NO:85), CDR2 having the amino acid sequenceYITNYGGSTYYPDTVKG (SEQ ID NO:86) and CDR3 having the amino acid sequenceQSNYDYDGWFAY (SEQ ID NO:87) and the light chain CDR sequences: CDR1having the amino acid sequence KASQEVGTAVA (SEQ ID NO:88), CDR2 havingthe amino acid sequence WASTRHT (SEQ ID NO:89) and CDR3 having the aminoacid sequence QQYSNYPLT (SEQ ID NO:90); and (b) two copies of a fusionprotein, each fusion protein comprising an effector attached to one copyof a dimerization and docking domain (DDD) moiety from a human proteinkinase A (PKA) regulatory subunit RIα, RIβ, RIIα or RIIβ; wherein theeffector is a cytokine or a F(ab′)₂, Fab, Fab′ or scFv from a secondantibody.
 16. The complex of claim 15, wherein the second antibody bindsto a tumor-associated antigen selected from the group consisting ofcarbonic anhydrase IX, CSAp (colon-specific antigen-p), CD1 (cluster ofdifferentiation 1), CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16,CD18, CD19, CD20, IGF-1R, CD21, CD22, CD23, CD25, CD29, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64,CD66a, CD66b, CD66c, CD66d, CD66e, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5 (carcinoembryonicantigen-related cell adhesion molecule 5), CEACAM6, ED-B (extradomain B)of fibronectin, Factor H, FHL-1 (four and a half LIM domains protein 1),Flt-3 (Fms-like tyrosine kinase 3), folate receptor, GRO-β (growthregulatory oncogene beta), HMGB-1 (high mobility group protein B1),hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1(ILGF-1), IFN-γ (interferon-gamma), IFN-α, IFN-β, IL-2 (interleukin-2),IL-4R (interleukin-4 receptor), IL-6R, IL-13R, IL-15R, IL-17R, IL-18R,IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10 (interferongamma-induced protein 10), MAGE (melanoma antigen), mCRP (modifiedC-reactive protein), MCP-1 (monocyte chemoattractant protein-1), MIP-1A(macrophage inflammatory protein-1A), MIP-1B, MIF (macrophage migrationinhibitory factor), MUC1 (mucin 1), MUC2, MUC3, MUC4, MUC5ac, NCA-95(normal glycoprotein crossreacting with CEA-95), NCA-90, PSMA(prostate-specific membrane antigen), EGP-1 (epithelial/carcinomaantigen-1), EGP-2, AFP (alpha fetoprotein), HLA-DR (human leukocyteantigen-DR), tenascin, Le(y) (Lewis antigen y), RANTES(regulated onactivation, normal T cell expressed and secreted), Tn (Thomsen-nouvelle)antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α(tumor necrosis factor-alpha), TRAIL (TNF-related apoptosis-inducingligand) receptor R1, TRAIL receptor R2, VEGFR (vascular endothelialgrowth factor receptor), EGFR (epidermal growth factor receptor), PlGF(placental growth factor), complement factors C3, complement factor C3a,complement factor C3b, complement factor C5a, complement factor C5, andan oncogene product.
 17. The complex of claim 15, wherein the cytokineis selected from the group consisting of human growth hormone,N-methionyl human growth hormone, bovine growth hormone, parathyroidhormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, thrombopoietin (TPO), NGF-β,platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I,insulin-like growth factor-II, erythropoietin (EPO), osteoinductivefactors, an interferon, interferon-α, interferon-β, interferon-β,macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-25, kit-ligand, angiostatin, thrombospondin, andendostatin.
 18. The complex of claim 15, wherein the cytokine isinterferon-α2b.
 19. The complex of claim 15, wherein the anti-IGF-1R orantigen-binding antibody fragment comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:94 and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:95.
 20. The complex of claim 15, wherein the anti-IGF-1Rantigen-binding antibody fragment comprises an Fc region capable ofinducing effector function, wherein the complex is effective in treatinga cancer that expresses human IGF-1R.
 21. The complex of claim 20,wherein the cancer is selected from the group consisting of Wilms'tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, aneuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer,rectal cancer, prostate cancer, liver cancer, renal cancer, pancreaticcancer, lung cancer, biliary cancer, cervical cancer, endometrialcancer, esophageal cancer, gastric cancer, head and neck cancer,medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia,myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin'slymphoma, non-Hodgkin's lymphoma, and urinary bladder cancer.
 22. Thecomplex of claim 20, wherein the cancer is renal cell carcinoma, breastcancer or pancreatic cancer.
 23. The complex of claim 15, furthercomprising a therapeutic agent selected from the group consisting of aradionuclide, an immunomodulator, an anti-angiogenic agent, a cytokine,a chemokine, a growth factor, a hormone, a drug, a prodrug, an enzyme, apro-apoptotic agent, a photoactive therapeutic agent, a cytotoxic agent,a chemotherapeutic agent and a toxin, wherein the therapeutic agent isconjugated to the complex.
 24. The complex of claim 23, wherein the drugis selected from the group consisting of 5-fluorouracil, azaribine,anastrozole, anthracyclines, bleomycin, bortezomib, bryostatin-1,busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, cladribine,camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel,dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine(2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, estramustine, epipodophyllotoxin, estrogenreceptor binding agents, etoposide (VP16), etoposide glucuronide,etoposide phosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR(FUdR-dO), fludarabine, flutamide, farnesyl-protein transferaseinhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, lenolidamide, leucovorin, lomustine, mechlorethamine,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, nitrosourea, plicomycin, procarbazine,paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin,tamoxifen, temazolomide, transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,vinblastine, vincristine and vinca alkaloids.
 25. The complex of claim23, wherein the toxin is selected from the group consisting of ricin,abrin, saporin, ribonuclease (RNase), ranpirnase DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.
 26. The complex ofclaim 23, wherein the radionuclide is selected from the group consistingof ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²,P, ³²P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh,¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.
 27. Acomposition comprising the complex of claim 15 and a buffer.